Peptides and compounds that bind to the IL-5 receptor

ABSTRACT

Interleukin-5 receptor (IL-5R) ligands comprising amino sequences of formula TGGGDGYVX 3 VE X 4  ARCPTCK, EGYVX 3 VE X 4  ARCPTCK, EGYVX 3 VE X 4  ARCPTCR, GYVX 3 VE X 4  ARCPTCG, EGYVX 3 VE X 4  ARCPTCG, GYVX 3 VE X 4  ARCPTCR, and EGYVX 3 VE X 4  AACPTCR, and methods of using the same, are described and claimed.

TECHNICAL FIELD

The present invention provides peptides and compounds that bind theinterleukin 5 receptors (IL-5R), methods for assaying interleukin 5(IL-5), and methods for inhibiting the binding of IL-5 to the IL-5R. Theinvention has application in the fields of biochemistry and medicinalchemistry and particularly provides IL-5 antagonists for use in thetreatment of human disease.

BACKGROUND ART

Interleukin-5 (IL-5 or IL5) is a lymphokine secreted by T cells and mastcells having biological activities on B cells and eosinophils. In murinehematopoiesis, IL-5 is a selective signal for the proliferation anddifferentiation of the eosinophilic lineage. See Yamaguchi et al., J.Exp. Med. 167:43–56 (1988). In this respect, IL-5 function showsanalogies with colony-stimulating factors for other myeloid lineages.Also, human (h) IL-5 is very potent in the activation of humaneosinophils. See Lopez et al., J. Exp. Med. 167:219–224 (1988) and Saitoet al., Proc. Natl. Acad. Sci. USA 85:2288–2292 (1988).

IL-5 mediates its activity through a cell membrane receptor-complex.This complex has been characterized physicochemically in both the murineand human system. Mouse pre-B cell lines depending on IL-5 for theirgrowth have been developed from bone marrow and are used for IL-5receptor analysis. See Rolink et al., J. Exp. Med. 169:1693–1701 (1989).The human IL-5 receptor can be studied on a subclone of thepromyelocytic cell line HL60 induced towards eosinophil differentiation.See Plaetinck et al., J. Exp. Med. 172:683–691 (1990).

Eosinophilic differentiation is initiated using sodium butyrate. Onlyhigh affinity (Kd=30 pM) IL-5 binding sites can be found on these cells.However, cross-linking studies reveal the presence of two polypeptidechains involved in IL-5 binding, i.e., IL-5R-α and IL-5R-β chains. Devoset al., Canadian Patent Publication 2,058,003 describes a recombinant αchain of human IL-5R or parts thereof, DNA-sequences coding for such areceptor or parts thereof, and host cells transformed with such vectors.Takatsu et al., European Patent Publication 475,746 provides an isolatedcDNA sequence coding for murine and human IL-5 receptor. Theextracellular domain (ECD) of the human IL-5R-α chain can be expressedin cells, such as CHO cells, in a manner that allows for the enzymaticharvest of the receptor from the cell surface and its subsequentimmobilization using a capture antibody (E. A. Whitehorn, et al.,Bio/Technology 13:1215 (1995).

A soluble human IL-5R-α chain can be used as an IL-5 antagonist inchronic asthma or other disease states with demonstrated eosinophilia.Eosinophils are white blood cells of the granulocytic lineage. Theirnormal function appears to be combating parasitic infections,particularly helminthis infections. However, their accumulation intissues, a condition referred to as eosinophilia, is also associatedwith several disease states, most notably asthma. It is believed thatthe damage to the epithelial lining of the bronchial passages in severeasthmatic attacks is largely caused by the compounds released bydegranulating eosinophils.

In U.S. Pat. No. 5,096,704, there is specifically disclosed the use ofcompounds which block the stimulatory effects of IL-5 in order toinhibit production and accumulation of eosinophils. The stimulatoryeffects of IL-5 were blocked by administering an effective amount of anantagonist to human interleukin-5, preferably using monoclonalantibodies or binding compositions derived therefrom by standardtechniques. Monoclonal antibodies were selected by their ability toinhibit IL-5 induced effects in standard IL-5 bioassays, such as theability to stimulate the growth and development of eosinophils in invitro colony forming assays, and the ability to augment in vitroproliferation of the in vivo passaged BCL1 lymphoma cells. The use ofantibody fragments, e.g., Fab fragments, was also reported.

U.S. Pat. Nos. 5,668,110; 5,677,280; 5,654,276, and 5,683,983 alsodiscuss peptides that bind the IL-5 receptors and block the effect ofIL-5.

Currently glucocorticoid steroids are the most effective drugs fortreating the acute effects of allergic diseases, such as asthma.However, the availability of alternative or complementary approaches tothe treatment of disorders associated with eosinophilia would haveimportant clinical utility.

Asthma has become the most common chronic disease in industrializedcountries. Conventional methods and therapeutic agents may not becompletely effective in the treatment of asthma or other immunomediatedinflammatory diseases in all patient populations. Moreover, thereremains a need for compounds that bind to or otherwise interact with theIL-5R, both for studies of the important biological activities mediatedby this receptor and for treatment of disease. The present inventionprovides such compounds.

DISCLOSURE OF THE INVENTION

This invention is directed, in part, to the novel and unexpecteddiscovery that defined low molecular weight peptides and peptidemimetics have strong binding properties to the IL-5 R. The peptides arefourteen to fifty or more amino acid residues in length, preferablyfourteen to twenty amino acid residues in length, and comprise a coresequence of amino acids selected from the following:

-   -   X₁-TGGGDGYVX₃VEX₄ARCPTCK-X₂ (residues 32–50 of SEQ ID NO: 1);    -   X₁-TGGGDGYVX₃VEX₄ARCPTCK-X₂ (residues 32–50 of SEQ ID NO: 1);    -   X₁-EGYVX₃VEX₄ARCPTCK-X₂ (residues 36–50 SEQ ID NO: 2);    -   X₁-EGYVX₃VEX₄ARCPTCR-X₂ (residues 36–50 SEQ ID NO: 3);    -   X₁-GYVX₃VEX₄ARCPTCG-X₂ (residues 36–50 SEQ ID NO: 4);    -   X₁-EGYVX₃VEX₄ARCPTCG-X₂ (residues 36–50 SEQ ID NO: 5);    -   X₁-GYVX₃VEX₄ARCPTCR-X₂ (residues 37–50 SEQ ID NO: 6); and    -   X₁-EGYVX₃VEX₄AACPTCR-X₂ (residues 36–50 SEQ ID NO: 7)        wherein X₁ is hydrogen or acyl; X₂ is —NH₂ or —OH wherein —NH₂        indicates that the carboxy terminus of the compound has been        amidated and —OH indicates that the carboxy terminus of the        compounds has not been derivatized; X₃ is Cys, Lys, or Dpr        wherein Dpr is diaminopropionic acid, and X₄ is Nal (where Nal        is 1-naphthylalanine), Trp, or Phe.

Particularly preferred compounds include the following.

-   -   (H)-TGGGDGYVCVEWARCPTCK-(OH) (residues 32–50 of SEQ ID NO: 8);    -   (Ac)-EGYV(Dpr)VEWARCPTCR-(NH₂) (residues 36–50 of SEQ ID NO: 9);    -   (Ac)-EGYVCVEWAACPTCR-(NH₂) (residues 36–50 of SEQ ID NO: 10);    -   (Ac)-EGYVCVEWARCPTCK-(NH₂) (residues 36–50 of SEQ ID NO: 11);    -   (Ac)-EGYVCVEWARCPTCK-(OH) (residues 36–50 of SEQ ID NO: 11);    -   (Ac)-EGYVCVEWARCPTCR-(NH₂) (residues 36–50 of SEQ ID NO: 12);    -   (Ahx)-EGYVCVEWARCPTCR-(NH₂) (residues 36–50 of SEQ ID NO: 12);    -   (H)-EGYVCVEFARCPTCG-(NH₂) (residues 36–50 of SEQ ID NO: 13);    -   (H)-EGYVCVEFARCPTCR-(OH) (residues 36–50 of SEQ ID NO: 14);    -   (H)-EGYVCVEWARCPTCG-(NH₂) (residues 36–50 of SEQ ID NO: 15);    -   (H)-EGYVCVEWARCPTCK-(NH₂) (residues 36–50 of SEQ ID NO: 11);    -   (H)-EGYVCVEWARCPTCK-(OH) (residues 36–50 of SEQ ID NO: 11);    -   (Ac)-EGYVCVEWARCPTCR-(OH) (residues 36–50 of SEQ ID NO: 12);    -   (H)-EGYVCVEWARCPTCR-(NH₂) (residues 36–50 of SEQ ID NO: 12);    -   (H)-EGYVCVEWARCPTCR-(OH) (residues 36–50 of SEQ ID NO: 12);    -   (H)-EGYVKVEWARCPTCR-(OH) (residues 36–50 of SEQ ID NO: 16);    -   (H)-GYVCVEFARCPTCG-(NH₂) (residues 37–50 of SEQ ID NO: 17); and    -   (H)-GYVCVEWARCPTCR-(OH) (residues 37–50 of SEQ ID NO: 18);        where —(NH₂) indicates that the carboxy terminus of the compound        has been amidated; where —(OH) indicates that the carboxy        terminus of the compound has not been derivatized; where (Ac)-        indicates that the amino terminus of the compound has been        acetylated; and where (Ahx)- indicates that the amino terminus        of the compound has been acylated with aminohexanoic acid.

Another embodiment is directed towards those compounds having anintramolecular disulfide linkage between two Cys residues.

More preferably, this invention provides dimers of the above sequences.These dimers can be formed via intermolecular disulfide, amide,carbamate, or urea linkages. The dimeric compounds may also containintramolecular cysteine linkages, as well.

Most preferably, the monomeric subunits will be dimerized to yieldcompounds having both intramolecular and intermolecular disulfide bondsas follows:

Preferably, the compound is covalently attached to one or more of avariety of hydrophilic polymers. The hydrophilic polymer(s) may beattached, for example, to one or both of the peptide chains in thedimeric compounds. If a hydrophilic polymer is attached to both peptidechains, the polymers may be the same or different, preferably they willbe the same. Preferably, such attachment is at the amino terminus of thecompound. It will be appreciated by those of skill in the art that whenthe compounds are attached to one or more of a variety of hydrophilicpolymers at the amino terminus (i.e., at X₁), then X₁ is the hydrophilicpolymer residue. The hydrophilic polymer has an average molecular weightof between about 500 to about 40,000 daltons, and more preferably,between about 5,000 and 20,000 daltons.

Preferably, the hydrophilic polymer is selected from the groupconsisting of polyethylene glycol (PEG), polypropylene glycol,polylactic acid, polyglycolic acid and copolymers thereof. Mostpreferably, the polymer is polyethylene glycol. A compound to which PEGis conjugated is herein termed “PEGylated.”

In a preferred embodiment, a peptide is conjugated to a PEG polymer,preferably at the amino terminus. The peptide may be a dimer comprisingPEGylated peptides.

The invention also provides for pharmaceutical compositions comprisingone or more of the compounds described herein and a physiologicallyacceptable carrier. These pharmaceutical compositions can be in avariety of forms including oral dosage forms, as well as inhalablepowders and solutions and injectable and infusible solutions.

Suitable pharmaceutically acceptable derivatives of the compoundsinclude pharmaceutically acceptable salts and acid addition salts,pharmaceutically acceptable esters, pharmaceutically acceptable amides,labeled compounds and compounds that are covalently attached to one ormore of a variety of hydrophilic polymers as defined hereinafter.

The peptides and peptide mimetics of the invention are useful fortherapeutic purposes in treating conditions mediated by IL-5 orinvolving improper production of or response to IL-5 and can be used toinhibit production and accumulation of eosinophils. These compounds willfind particular use in the treatment of asthma. Thus, the presentinvention also provides a method for treating a patient having adisorder that is susceptible to treatment with a IL-5 inhibitor, whereinthe patient receives, or is administered, a therapeutically effectivedose or amount of a compound of the present invention.

A further aspect of the invention is drawn to a methods for treatingconditions mediated by IL-5 or involving improper production of orresponse to IL-5 or for inhibiting production and accumulation ofeosinophils comprising the steps of administering a compound thateffects homodimerization of the alpha chain of the IL-5 receptor, thuspreventing alpha chain participation in the IL-5/alpha chain/beta chaincomplex required for IL-5 signal transduction. Preferably, thesecompounds are dimeric in structure wherein each monomeric subunit iscapable of binding to the alpha chain of the IL-5 receptor.

Peptides and peptide mimetics suitable for therapeutic and/or diagnosticpurposes have an IC₅₀ of about 2 mM or less, as determined by thebinding affinity assay set forth below wherein a lower IC₅₀ correlatesto a stronger binding affinity to IL-5R. For pharmaceutical purposes,the peptides and peptidomimetics preferably have an IC₅₀ of no more thanabout 100 μM.

When used for diagnostic purposes, the peptides and peptide mimeticspreferably are labeled with a detectable label and, accordingly, thepeptides and peptide mimetics without such a label serve asintermediates in the preparation of labeled peptides and peptidemimetics.

Peptides meeting the defined criteria for molecular weight and bindingaffinity for IL-5R comprise 15 or more amino acids wherein the aminoacids are naturally occurring or synthetic (non-naturally occurring)amino acids. Peptide mimetics include peptides having one or more of thefollowing modifications:

-   -   peptides wherein one or more of the peptidyl [—C(O)NR—] linkages        (bonds) have been replaced by a non-peptidyl linkage such as a        —CH₂-carbamate linkage [—CH₂—OC(O)NR—]; a phosphonate linkage; a        —CH₂-sulfonamide [—CH₂—S(O)2NR—] linkage; a urea [—NHC(O)NH—]        linkage; a —CH₂-secondary amine linkage; or an alkylated        peptidyl linkage [—C(O)NR₆— where R₆ is lower alkyl];    -   peptides wherein the N-terminus is derivatized to a —NRR₁ group;        to a —NRC(O)R group; to a —NRC(O)OR group; to a —NRS(O)₂R group;        to a —NHC(O)NHR group where R and R₁ are hydrogen or lower alkyl        with the proviso that R and R₁ are not both hydrogen; to a        succinimide group; to a benzyloxycarbonyl-NH— (CBZ-NH—) group;        or to a benzyloxycarbonyl-NH— group having from 1 to 3        substituents on the phenyl ring selected from the group        consisting of lower alkyl, lower alkoxy, chloro, and bromo; or    -   peptides wherein the C terminus is derivatized to —C(O)R₂ where        R₂ is selected from the group consisting of lower alkoxy, and        —NR₃R₄ where R₃ and R₄ are independently selected from the group        consisting of hydrogen and lower alkyl.

Accordingly, preferred peptides and peptide mimetics comprise a compoundhaving a binding affinity to IL-5R as expressed by an IC₅₀ of no morethan about 100 μM,

-   -   wherein from zero to all of the —C(O)NH— linkages of the peptide        have been replaced by a linkage selected from the group        consisting of a —CH₂OC(O)NR— linkage; a phosphonate linkage; a        —CH₂S(O)₂NR— linkage; a —CH₂NR— linkage; and a —C(O)NR₆—        linkage; and a —NHC(O)NH— linkage where R is hydrogen or lower        alkyl and R₆ is lower alkyl,    -   further wherein the N-terminus of said peptide or peptide        mimetic is selected from the group consisting of a —NRR₁ group;        a —NRC(O)R group; a —NRC(O)OR group; a —NRS(O)₂R group; a        —NHC(O)NHR group; a succinimide group; a benzyloxycarbonyl-NH—        group; and a benzyloxycarbonyl-NH— group having from 1 to 3        substituents on the phenyl ring selected from the group        consisting of lower alkyl, lower alkoxy, chloro, and bromo,        where R and R₁ are independently selected from the group        consisting of hydrogen and lower alkyl, and still further        wherein the C-terminus of said peptide or peptide mimetic has        the formula —C(O)R₂ where R₂ is selected from the group        consisting of hydroxy, lower alkoxy, and —NR₃R₄ where R₃ and R₄        are independently selected from the group consisting of hydrogen        and lower alkyl and where the nitrogen atom of the —NR₃R₄ group        can optionally be the amine group of the N-terminus of the        peptide so as to form a cyclic peptide, and physiologically        acceptable salts thereof.

In a related embodiment, the invention is directed to a labeled peptideor peptide mimetic comprising a peptide or peptide mimetic described asabove having covalently attached thereto a label capable of detection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a competitive binding experimentdemonstrating blockade of IL-5 binding by AF18748.

FIG. 2 shows the results of a functional binding experimentdemonstrating inhibition of IL-5-induced eosinophil adhesion by AF18748.

FIG. 3 shows the specificity of action of AF18748, which has no effecton eosinophil adhesion induced by GM-CSF, IL-3, TNFα or fMet-Leu-Phe.

FIG. 4 shows fluorescence as a function of the concentration of AF18748(closed circles), AF17121 (open circles) or human IL-5 (open squares)added to microtiter plates containing IL-5RαECD-coated beads.

FIG. 5 shows a gel filtration chromatograph where IL-5RαECD (dashedline), IL5RαECD plus AF17121 (dotted line) or IL-5RαECD plus AF18748(solid line).

FIG. 6 shows the results of velocity sedimentation experiments withIL-5RαECD (3.3S) and IL-5RαECD in the presence of AF18748: another peakappears at 5.1S indicating dimerization.

FIG. 7 shows the response of cells expressing chimeric IL-5Rα/EGFRtreated with AF18748 (open circles), AF17121 (closed circles) or IL-5(open squares).

FIG. 8 shows the response of cells expressing chimeric IL-5Rα/EGFRtreated with 200 pM AF18748 in the presence of increasing amounts ofIL-5.

FIG. 9 shows the stability of AF18748 (closed circles), AF25123 (closedupward-pointing triangles) and AF25122 (closed downward-pointingtriangles) in vivo.

MODES FOR CARRYING OUT THE INVENTION

Definitions and Nomenclature

The following definitions are set forth to illustrate and define themeaning and scope of the various terms used to describe the inventionherein.

“Pharmaceutically acceptable salts” refer to the non-toxic alkali metal,alkaline earth metal, and ammonium salts commonly used in thepharmaceutical industry including the sodium, potassium, lithium,calcium, magnesium, barium, ammonium, and protamine zinc salts, whichare prepared by methods well known in the art. The term also includesnon-toxic acid addition salts, which are generally prepared by reactingthe compounds of this invention with a suitable organic or inorganicacid. Representative salts include the hydrochloride, hydrobromide,sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, napsylate, and the like.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases and which are not biologically or otherwise undesirable, formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, menthanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. For a descriptionof pharmaceutically acceptable acid addition salts as prodrugs, seeBundgaard, H., ed., (1985) Design of Prodrugs, Elsevier SciencePublishers, Amsterdam.

“Pharmaceutically acceptable ester” refers to those esters which retain,upon hydrolysis of the ester bond, the biological effectiveness andproperties of the carboxylic acid or alcohol and are not biologically orotherwise undesirable. For a description of pharmaceutically acceptableesters as prodrugs, see Bundgaard, H., supra. These esters are typicallyformed from the corresponding carboxylic acid and an alcohol. Generally,ester formation can be accomplished via conventional synthetictechniques. (See, e.g., March, Advanced Organic Chemistry, 3rd Ed., JohnWiley & Sons, New York (1985) p. 1157 and references cited therein, andMark et al., Encyclopedia of Chemical Technology, John Wiley & Sons, NewYork (1980).) The alcohol component of the ester will generally comprise(i) a C₂–C₁₂ aliphatic alcohol that can or can not contain one or moredouble bonds and can or can not contain branched carbon chains or (ii) aC₇–C₁₂ aromatic or heteroaromatic alcohols. This invention alsocontemplates the use of those compositions which are both esters asdescribed herein and at the same time are the pharmaceuticallyacceptable acid addition salts thereof.

“Pharmaceutically acceptable amide” refers to those amides which retain,upon hydrolysis of the amide bond, the biological effectiveness andproperties of the carboxylic acid or amine and are not biologically orotherwise undesirable. For a description of pharmaceutically acceptableamides as prodrugs, see Bundgaard, H., ed., supra. These amides aretypically formed from the corresponding carboxylic acid and an amine.Generally, amide formation can be accomplished via conventionalsynthetic techniques. (See, e.g., March, Advanced Organic Chemistry, 3rdEd., John Wiley & Sons, New York (1985) p. 1152 and Mark et al.,Encyclopedia of Chemical Technology, John Wiley & Sons, New York(1980).) This invention also contemplates the use of those compositionswhich are both amides as described herein and at the same time are thepharmaceutically acceptable acid addition salts thereof.

“Pharmaceutically or therapeutically acceptable carrier” refers to acarrier medium which does not interfere with the effectiveness of thebiological activity of the active ingredients and which is not toxic tothe host or patient.

“Stereoisomer” refers to a chemical compound having the same molecularweight, chemical composition, and constitution as another, but with theatoms grouped differently. That is, certain identical chemical moietiesare at different orientations in space and, therefore, when pure, hasthe ability to rotate the plane of polarized light. However, some purestereoisomers may have an optical rotation that is so slight that it isundetectable with present instrumentation. The compounds of the instantinvention may have one or more asymmetrical carbon atoms and thereforeinclude various stereoisomers. All stereoisomers are included within thescope of the invention.

“Therapeutically or pharmaceutically effective amount” as applied to thecompositions of the instant invention refers to the amount ofcomposition sufficient to induce a desired biological result. Thatresult can be alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. In thepresent invention, the result will typically involve a decrease in theimmunological and/or inflammatory responses to infection or tissueinjury.

Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn orN; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Gluor E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg orR; and Glycine is Gly or G. In addition, the abbreviation Nal is used todenote 1-naphthylalanine

In addition to peptides consisting only of naturally-occurring aminoacids, peptidomimetics or peptide analogs are also provided. Peptideanalogs are commonly used in the pharmaceutical industry as non-peptidedrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics” (Fauchere, J., Adv. Drug Res. 15:29 (1986); Veber andFreidinger, TINS p. 392 (1985); and Evans et al., J Med. Chem. 30:1229(1987), which are incorporated herein by reference). Peptide mimeticsthat are structurally similar to therapeutically useful peptides may beused to produce an equivalent or enhanced therapeutic or prophylacticeffect. Generally, peptidomimetics are structurally similar to aparadigm polypeptide (i.e., a polypeptide that has a biological orpharmacological activity), such as naturally-occurring receptor-bindingpolypeptide, but have one or more peptide linkages optionally replacedby a linkage selected from the group consisting of: —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH—(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, bymethods known in the art and further described in the followingreferences: Spatola, A. F. in Chemistry and Biochemistry of Amino Acids,Peptides and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p.267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3,PEPTIDE BACKBONE MODIFICATIONS (general review); Morley, Trends PharmSci (1980) pp. 463–468 (general review); Hudson, D. et al., Int J PeptProt Res 14:177–185 (1979): (—CH₂NH—, CH₂CH₂—); Spatola et al., Life Sci38:1243–1249 (1986): (—CH₂—S); Hann J. Chem. Soc. Perkin Trans. I307–314 (1982):(—CH—CH—, cis and trans); Almquist et al., J Med Chem23:1392–1398 (1980): (—COCH₂—); Jennings-White et al., Tetrahedron Lett23:2533 (1982): (—COCH₂—); Szelke et al., European Application. EP 45665CA: 97:39405 (1982) (—CH(OH)CH₂—); Holladay et al., Tetrahedron Lett24:4401–4404 (1983): (—C(OH)CH₂—); and Hruby Life Sci 31:189–199 (1982):(—CH₂—S—); each of which is incorporated herein by reference. Aparticularly preferred non-peptide linkage is —CH₂NH—. Such peptidemimetics may have significant advantages over polypeptide embodiments,including, for example: more economical production, greater chemicalstability, enhanced pharmacological properties (half-life, absorption,potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum ofbiological activities), reduced antigenicity, and others. Labeling ofpeptidomimetics usually involves covalent attachment of one or morelabels, directly or through a spacer (e.g., an amide group), tonon-interfering position(s) on the peptidomimetic that are predicted byquantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions generally are positions that do not formdirect contacts with the macromolecules(s) (e.g., immunoglobulinsuperfamily molecules) to which the peptidomimetic binds to produce thetherapeutic effect. Derivatization (e.g., labeling) of peptidomimeticsshould not substantially interfere with the desired biological orpharmacological activity of the peptidomimetic. Generally,peptidomimetics of receptor-binding peptides bind to the receptor withhigh affinity and possess detectable biological activity (i.e., areagonistic or antagonistic to one or more receptor-mediated phenotypicchanges).

Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used to generate more stable peptides. For theavoidance of doubt, the abbreviation

whenever used herein is meant to indicate the structure wherein the sidechain amino residues of each lysine are coupled via a urea linkage.Overview

The present invention provides compounds that bind to the IL-5R. Thesecompounds include “lead” peptide compounds and “derivative” compoundsconstructed so as to have the same or similar molecular structure orshape as the lead compounds but that differ from the lead compoundseither with respect to susceptibility to hydrolysis or proteolysisand/or with respect to other biological properties, such as increasedaffinity for the receptor. The present invention also providescompositions comprising an effective IL-5R binding, IL-5 blockingcompound, and more particularly a compound, that is useful for treatingdisorders associated with the overexpression of IL-5 or with theproduction and accumulation of eosinophils.

Identification of Peptides which Bind IL-5R

A. The Receptor

The immobilized α chain, β chain, and heterodimer, as well as theextracellular domains of the single chains of the IL-5 receptors wereproduced in recombinant host cells. The DNA encoding IL-5R was obtainedby PCR of cDNA from TF-1 cells using primers obtained from the publishedreceptor sequences. See Murata (1992) J. Exp. Med. 175:341–351 andHayashida (1990) Proc. Natl. Acad. Sci. USA 87:9655–9659, each of whichis incorporated herein by reference. One useful form of IL-5R isconstructed by expressing the protein as a soluble protein inbaculovirus transformed host cells using standard methods; anotheruseful form is constructed with a signal peptide for protein secretionand for glycophospholipid membrane anchor attachment. This form ofanchor attachment is called “PIG-tailing.” See Caras and Wendell,Science 243: 1196–1198 (1989) and Lin et al., Science 249:677–679(1990).

Using the PIG-tailing system, one can cleave the receptor from thesurface of the cells expressing the receptor (e.g., transformed CHOcells selected for high level expression of receptor with a cell sorter)with phospholipase C. The cleaved receptor still comprises a carboxyterminal sequence of amino acids, called the “HPAP tail,” from thesignal protein for membrane attachment and can be immobilized withoutfurther purification. The recombinant receptor protein can beimmobilized by coating the wells of microtiter plates with an anti-HPAPtail antibody (Ab 179), blocking non-specific binding with bovine serumalbumin (BSA) in PBS, and then binding cleaved recombinant receptor tothe antibody. Using this procedure, one should perform theimmobilization reaction in varying concentrations of receptor todetermine the optimum amount for a given preparation, because differentpreparations of recombinant protein often contain different amounts ofthe desired protein. See U.S. patent application Ser. No. 07/947,339,filed Sep. 18, 1992, incorporated herein by reference.

To discriminate among higher affinity peptides, a monovalent receptorprobe frequently is used. This probe can be produced using proteinkinase A to phosphorylate a peptide sequence fused to the C-terminus ofthe soluble receptor. The “engineered” form of the IL-5 receptor a chainand β chain are then expressed in host cells, typically CHO cells.Following PI-PLC harvest of the receptors, the receptor is labeled tohigh specific activity with ³³P or ³²P for use as a monovalent probe toidentify high affinity ligands using colony lifts.

B. The Peptides

Preferred methods to facilitate identification of peptides which bindIL-5R involve first identifying lead peptides which bind the receptorand then making other peptides which resemble the lead peptides.

A representative listing of preferred compounds of the invention isshown in Tables 1 and 2 below.

TABLE 1 X₁-TGGGDGYVX₃VEX₄ARCPTCK-X₂ (residues 32–50 of SEQ ID NO: 1);X₁-EGYVX₃VEX₄ARCPTCK-X₂ (residues 36–50 of SEQ ID NO: 2);X₁-EGYVX₃VEX₄ARCPTCR-X₂ (residues 36–50 of SEQ ID NO: 3); X₁-GYVX₃VEX₄ARCPTCG-X₂ (residues 36–50 of SEQ ID NO: 4);X₁-EGYVX₃VEX₄ARCPTCG-X₂ (residues 36–50 of SEQ ID NO: 5);X₁-GYVX₃VEX₄ARCPTCR-X₂ (residues 37–50 of SEQ ID NO: 6); andX₁-EGYVX₃VEX₄AACPTCR-X₂ (residues 36–50 of SEQ ID NO: 7)wherein X₁ is hydrogen or acyl; X₂ is —NH₂ or —OH wherein —NH₂ indicatesthat the carboxy terminus of the compound has been amidated and —OHindicates that the carboxy terminus of the compounds has not beenderivatized; X₃ is Cys, Lys, or Dpr wherein Dpr is diaminopropionicacid, and X₄ is Nal, Trp, or Phe. Another embodiment is directed towardsthose compounds having an intramolecular disulfide linkage betweencysteine residues.

Particularly preferred compounds include the following.

-   -   (H)-TGGGDGYVCVEWARCPTCK-(OH) (residues 32–50 of SEQ ID NO: 8);    -   (Ac)-EGYV(Dpr)VEWARCPTCR-(NH₂) (residues 36–50 of SEQ ID NO: 9);    -   (Ac)-EGYVCVEWAACPTCR-(NH₂) (residues 36–50 of SEQ ID NO: 10);    -   (Ac)-EGYVCVEWARCPTCK-(NH₂) (residues 36–50 of SEQ ID NO: 11);    -   (Ac)-EGYVCVEWARCPTCK-(OH) residues 36–50 of (SEQ ID NO: 11);    -   (Ac)-EGYVCVEWARCPTCR-(NH₂) (residues 36–50 of SEQ ID NO: 12);    -   (Ahx)-EGYVCVEWARCPTCR-(NH₂) (residues 36–50 of SEQ ID NO: 12);    -   (H)-EGYVCVEFARCPTCG-(NH₂) (residues 36–50 of SEQ ID NO: 13);    -   (H)-EGYVCVEFARCPTCR-(OH) (residues 36–50 of SEQ ID NO: 14);    -   (H)-EGYVCVEWARCPTCG-(NH₂) (residues 36–50 of SEQ ID NO: 15);    -   (H)-EGYVCVEWARCPTCK-(NH₂) (residues 36–50 of SEQ ID NO: 11);    -   (H)-EGYVCVEWARCPTCK-(OH) (residues 36–50 of SEQ ID NO: 11);    -   (Ac)-EGYVCVEWARCPTCR-(OH) (residues 36–50 of SEQ ID NO: 12);    -   (H)-EGYVCVEWARCPTCR-(NH₂) (residues 36–50 of SEQ ID NO: 12);    -   (H)-EGYVCVEWARCPTCR-(OH) (residues 36–50 of SEQ ID NO: 12);    -   (H)-EGYVKVEWARCPTCR-(OH) (residues 36–50 of SEQ ID NO: 16);    -   (H)-GYVCVEFARCPTCG-(NH₂) (residues 37–50 of SEQ ID NO: 17); and    -   (H)-GYVCVEWARCPTCR-(OH) (residues 37–50 of SEQ ID NO: 18);        where —(NH₂) indicates that the carboxy terminus of the compound        has been amidated; where —(OH) indicates that the carboxy        terminus of the compound has not been derivatized; where (Ac)-        indicates that the amino terminus of the compound has been        acetylated; and where (Ahx)- indicates that the amino terminus        of the compound has been acylated with aminohexanoic acid.

More preferably, this invention provides dimers of the above sequences.For example, a sidechain primary amino group of one monomeric subunitcan be coupled to a sidechain primary amino group of a second monomericsubunit via a urea linkage. Likewise, one of skill in the art willreadily appreciate that side chain functionality of one monomericsubunit can be coupled to side chain functionality of a second subunitthrough a variety of strategies, including without limitation,intermolecular disulfide, ester, amide, carbamate, or urea linkages.

Particularly preferred compounds include disulfide linked dimers of theabove compounds which also have one or more intramolecular disulfidebonds. Most preferred are dimers having the following pattern ofdisulfide linkages:

C. Affinity

A variety of methods can be used to evaluate IC₅₀ values. For example,binding assays were used to determine whether the peptides inhibit thebinding of IL-5 to the extracellular domain of the IL-5 receptorα-chain. Alternatively, for some peptides, a microphysiometer assay wasused to determine whether the peptide blocked the response of TF-1 cellsto IL-5 (5 ng/ml).

To determine whether these peptides had any effect on IL-5 mediatedsignal transduction, they were tested for IL-5R agonist and antagonistactivity in a microphysiometer assay using the IL-5 responsive humanleukemia cell line, TF-1. Following overnight IL-5 starvation, thesecells exhibit a rapid and robust increase in metabolic activity uponaddition of IL-5 to the cell culture medium. A preferred compound wastested in the TF-1 cell microphysiometer assay and found to almostcompletely block the response of the cells to 400 pM IL-5 when tested at10 μM concentration. The peptide was of sufficiently high affinity toallow us to determine an accurate microphysiometer assay IC₅₀ value.

Typically, the IC₅₀ values were determined using the free peptide,although in some instances, it may be preferable to amidate theC-terminus or to prepare an ester or other carboxy amide. The N-terminaland C-terminal amino acids of the synthetic peptides are often precededby one or two glycine residues. These glycines are not believed to benecessary for binding or activity.

Peptides and peptidomimetics having an IC₅₀ of greater than about 100 mMlack sufficient binding to permit use in either the diagnostic ortherapeutic aspects of this invention. Preferably, for diagnosticpurposes, the peptides and peptidomimetics have an IC₅₀ of about 2.5 mMor less and, for pharmaceutical purposes, the peptides andpeptidomimetics have an IC₅₀ of about 2 mM or less.

IV. Preparation of Peptides and Peptide Mimetics

The peptides of the invention can be prepared by classical methods knownin the art, for example, by using standard solid phase techniques. Thestandard methods include exclusive solid phase synthesis, partial solidphase synthesis methods, fragment condensation, classical solutionsynthesis, and even by recombinant DNA technology. See, e.g., Merrifield(1963) J. Am. Chem. Soc. 85:2149. On solid phase, the synthesis istypically commenced from the C-terminal end of the peptide using analpha-amino protected resin. A suitable starting material can beprepared, for instance, by attaching the required alpha-amino acid to achloromethylated resin, a hydroxymethyl resin, or a benzhydrylamineresin. One such chloromethylated resin is sold under the trade nameBIO-BEADS SX-1 by Bio Rad Laboratories, Richmond, Calif., and thepreparation of the hydroxymethyl resin is described by Bodonszky et al.,Chem. Ind. (London) 38:1597 (1966). The benzhydrylamine (BHA) resin hasbeen described by Pietta and Marshall, Chem. Commn. 650 (1970), and iscommercially available from Beckman Instruments, Inc., Palo Alto,Calif., in the hydrochloride form.

Thus, the compounds of the invention can be prepared by coupling analpha-amino protected amino acid to the chloromethylated resin with theaid of, for example, cesium bicarbonate catalyst, according to themethod described by Gisin (1973) Helv. Chim. Acta 56:1467. After theinitial coupling, the alpha-amino protecting group is removed by achoice of reagents including trifluoroacetic acid (TFA) or hydrochloricacid (HCl) solutions in organic solvents at room temperature.

The alpha-amino protecting groups are those known to be useful in theart of stepwise synthesis of peptides. Included are acyl type protectinggroups (e.g. formyl, trifluoroacetyl, acetyl), aromatic urethane typeprotecting groups (e.g. benzyloxycarboyl (Cbz) and substituted Cbz),aliphatic urethane protecting groups (e.g. t-butyloxycarbonyl (Boc),isopropyloxycarbonyl, cyclohexyloxycarbonyl) and alkyl type protectinggroups (e.g. benzyl, triphenylmethyl, and fluorenylmethyl oxycarbonyl(Fmoc)). Boc and Fmoc are preferred protecting groups. The side chainprotecting group remains intact during coupling and is not split offduring the deprotection of the amino-terminus protecting group or duringcoupling. The side chain protecting group must be removable upon thecompletion of the synthesis of the final peptide and under reactionconditions that will not alter the target peptide.

The side chain protecting groups for Tyr include tetrahydropyranyl,tert-butyl, trityl, benzyl, Cbz, Z-Br-Cbz, and 2,5-dichlorobenzyl. Theside chain protecting groups for Asp include benzyl, 2,6-dichlorobenzyl,methyl, ethyl, and cyclohexyl. The side chain protecting groups for Thrand Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl,2,6-dichlorobenzyl, and Cbz. The side chain protecting group for Thr andSer is benzyl. The side chain protecting groups for Arg include nitro,Tosyl (Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), or Boc.The side chain protecting groups for Lys include Cbz,2-chlorobenzyloxycarbonyl (2-Cl-Cbz), 2-bromobenzyloxycarbonyl(2-BrCbz), Tos, or Boc.

After removal of the alpha-amino protecting group, the remainingprotected amino acids are coupled stepwise in the desired order. Anexcess of each protected amino acid is generally used with anappropriate carboxyl group activator such as dicyclohexylcarbodiimide(DCC) in solution, for example, in methylene chloride (CH₂Cl₂), dimethylformamide (DMF) mixtures.

After the desired amino acid sequence has been completed, the desiredpeptide is decoupled from the resin support by treatment with a reagentsuch as trifluoroacetic acid or hydrogen fluoride (HF), which not onlycleaves the peptide from the resin, but also cleaves all remaining sidechain protecting groups. When the chloromethylated resin is used,hydrogen fluoride treatment results in the formation of the free peptideacids. When the benzhydrylamine resin is used, hydrogen fluoridetreatment results directly in the free peptide amide. Alternatively,when the chloromethylated resin is employed, the side chain protectedpeptide can be decoupled by treatment of the peptide resin with ammoniato give the desired side chain protected amide or with an alkylamine togive a side chain protected alkylamide or dialkylamide. Side chainprotection is then removed in the usual fashion by treatment withhydrogen fluoride to give the free amides, alkylamides, ordialkylamides.

These solid phase peptide synthesis procedures are well known in the artand further described in Stewart, Solid Phase Peptide Syntheses (Freemanand Co., San Francisco, 1969).

In addition, in some preferred embodiments of the invention having oneor more cysteine residues in the compound, one or more of the cysteinesmay bear an appropriate protecting group, preferably an Acm group, toprevent or hinder undesired disulfide formation.

These solid phase peptide synthesis procedures are well known in the artand further described in Stewart, Solid Phase Peptide Syntheses (Freemanand Co., San Francisco, 1969). These procedures can also be used tosynthesize peptides in which amino acids other than the 20 naturallyoccurring, genetically encoded amino acids are substituted at one, two,or more positions of any of the compounds of the invention. Forinstance, naphthylalanine can be substituted for tryptophan,facilitating synthesis. Other synthetic amino acids that can besubstituted into the peptides of the present invention includeL-hydroxypropyl, L-3, 4-dihydroxyphenylalanine, α amino acids such asL-α-hydroxylysine and D-α-methylalanine, L-α methylalanine, β aminoacids, and isoquinoline. D amino acids and non-naturally occurringsynthetic amino acids can also be incorporated into the peptides of thepresent invention.

One can replace the naturally occurring side chains of the 20genetically encoded amino acids (or D amino acids) with other sidechains, for instance with groups such as alkyl, lower alkyl, cyclic 4-,5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(loweralkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivativesthereof, and with 4-, 5-, 6-, to 7-membered heterocyclic. In particular,proline analogs in which the ring size of the proline residue is changedfrom 5 members to 4, 6, or 7 members can be employed. Cyclic groups canbe saturated or unsaturated, and if unsaturated, can be aromatic ornon-aromatic.

The peptides typically are synthesized as the free acid but, as notedabove, could be readily prepared as the amide (i.e., designated with a—(NH₂) at the carboxy terminus of the compound) or ester. One can alsomodify the amino and/or carboxy terminus of the peptide compounds of theinvention to produce other compounds of the invention. Amino terminusmodifications include methylating (i.e., —NHCH₃ or —NH(CH₃)₂),acetylating, adding a carbobenzoyl group, or blocking the amino terminuswith any blocking group containing a carboxylate functionality definedby RCOO—, where R is selected from the group consisting of naphthyl,acridinyl, steroidyl, and similar groups. Carboxy terminus modificationsinclude replacing the free acid with a carboxamide group or forming acyclic lactam at the carboxy terminus to introduce structuralconstraints.

Amino terminus modifications are as recited above and includealkylating, acetylating, adding a carbobenzoyl group, forming asuccinimide group, etc. Specifically, the N-terminal amino group canthen be reacted as follows:

-   -   (a) to form an amide group of the formula RC(O)NH— where R is as        defined above by reaction with an acid halide [e.g., RC(O)Cl] or        acid anhydride. Typically, the reaction can be conducted by        contacting about equimolar or excess amounts (e.g., about 5        equivalents) of an acid halide to the peptide in an inert        diluent (e.g., dichloromethane) preferably containing an excess        (e.g., about 10 equivalents) of a tertiary amine, such as        diisopropylethylamine, to scavenge the acid generated during        reaction. Reaction conditions are otherwise conventional (e.g.,        room temperature for 30 minutes). Alkylation of the terminal        amino to provide for a lower alkyl N-substitution followed by        reaction with an acid halide as described above will provide for        N-alkyl amide group of the formula RC(O)NR—;    -   (b) to form a succinimide group by reaction with succinic        anhydride. As before, an approximately equimolar amount or an        excess of succinic anhydride (e.g., about 5 equivalents) can be        employed and the amino group is converted to the succinimide by        methods well known in the art including the use of an excess        (e.g., ten equivalents) of a tertiary amine such as        diisopropylethylamine in a suitable inert solvent (e.g.,        dichloromethane). See, for example, Wollenberg et al., U.S. Pat.        No. 4,612,132 which is incorporated herein by reference in its        entirety. It is understood that the succinic group can be        substituted with, for example, C₂–C₆ alkyl or —SR substituents        which are prepared in a conventional manner to provide for        substituted succinimide at the N-terminus of the peptide. Such        alkyl substituents are prepared by reaction of a lower olefin        (C₂–C₆) with maleic anhydride in the manner described by        Wollenberg et al., and —SR substituents are prepared by reaction        of RSH with maleic anhydride where R is as defined above;    -   (c) to form a benzyloxycarbonyl-NH— or a substituted        benzyloxycarbonyl-NH— group by reaction with approximately an        equivalent amount or an excess of CBZ-Cl (i.e.,        benzyloxycarbonyl chloride) or a substituted CBZ-Cl in a        suitable inert diluent (e.g., dichloromethane) preferably        containing a tertiary amine to scavenge the acid generated        during the reaction;    -   (d) to form a sulfonamide group by reaction with an equivalent        amount or an excess (e.g., 5 equivalents) of R—S(O)₂Cl in a        suitable inert diluent (dichloromethane) to convert the terminal        amine into a sulfonamide where R is as defined above.        Preferably, the inert diluent contains excess tertiary amine        (e.g., ten equivalents) such as diisopropylethylamine, to        scavenge the acid generated during reaction. Reaction conditions        are otherwise conventional (e.g., room temperature for 30        minutes);    -   (e) to form a carbamate group by reaction with an equivalent        amount or an excess (e.g., 5 equivalents) of R—OC(O)Cl or        R—OC(O)OC₆H₄-p-NO₂ in a suitable inert diluent (e.g.,        dichloromethane) to convert the terminal amine into a carbamate        where R is as defined above. Preferably, the inert diluent        contains an excess (e.g., about 10 equivalents) of a tertiary        amine, such as diisopropylethylamine, to scavenge any acid        generated during reaction. Reaction conditions are otherwise        conventional (e.g., room temperature for 30 minutes); and    -   (f) to form a urea group by reaction with an equivalent amount        or an excess (e.g., 5 equivalents) of R—N═C═O in a suitable        inert diluent (e.g., dichloromethane) to convert the terminal        amine into a urea (i.e., RNHC(O)NH—) group where R is as defined        above. Preferably, the inert diluent contains an excess (e.g.,        about 10 equivalents) of a tertiary amine, such as        diisopropylethylamine. Reaction conditions are otherwise        conventional (e.g., room temperature for about 30 minutes).

In preparing peptide mimetics wherein the C-terminal carboxyl group isreplaced by an ester (i.e., —C(O)OR where R is as defined above), theresins used to prepare the peptide acids are employed, and the sidechain protected peptide is cleaved with base and the appropriatealcohol, e.g., methanol. Side chain protecting groups are then removedin the usual fashion by treatment with hydrogen fluoride to obtain thedesired ester.

In preparing peptide mimetics wherein the C-terminal carboxyl group isreplaced by the amide —C(O)NR₃R₄, a benzhydrylamine resin is used as thesolid support for peptide synthesis. Upon completion of the synthesis,hydrogen fluoride treatment to release the peptide from the supportresults directly in the free peptide amide (i.e., the C-terminus is—C(O)NH₂). Alternatively, use of the chloromethylated resin duringpeptide synthesis coupled with reaction with ammonia to cleave the sidechain protected peptide from the support yields the free peptide amideand reaction with an alkylamine or a dialkylamine yields a side chainprotected alkylamide or dialkylamide (i.e., the C-terminus is —C(O)NRR1where R and R1 are as defined above). Side chain protection is thenremoved in the usual fashion by treatment with hydrogen fluoride to givethe free amides, alkylamides, or dialkylamides.

In another alternative embodiment, the C-terminal carboxyl group or aC-terminal ester can be induced to cyclize by internal displacement ofthe —OH or the ester (—OR) of the carboxyl group or ester respectivelywith the N-terminal amino group to form a cyclic peptide. For example,after synthesis and cleavage to give the peptide acid, the free acid isconverted to an activated ester by an appropriate carboxyl groupactivator such as dicyclohexylcarbodiimide (DCC) in solution, forexample, in methylene chloride (CH₂Cl₂), dimethyl formamide (DMF)mixtures. The cyclic peptide is then formed by internal displacement ofthe activated ester with the N-terminal amine. Internal cyclization asopposed to polymerization can be enhanced by use of very dilutesolutions. Such methods are well known in the art.

One can also cyclize the peptides of the invention, or incorporate adesamino or descarboxy residue at the termini of the peptide, so thatthere is no terminal amino or carboxyl group, to decrease susceptibilityto proteases or to restrict the conformation of the peptide. C-terminalfunctional groups of the compounds of the present invention includeamide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,and carboxy, and the lower ester derivatives thereof, and thepharmaceutically acceptable salts thereof.

One can also readily modify peptides by phosphorylation, and othermethods for making peptide derivatives of the compounds of the presentinvention are described in Hruby et al., Biochem J 268 (2):249–262(1990), incorporated herein by reference. Thus, the peptide compounds ofthe invention also serve as structural models for non-peptidic compoundswith similar biological activity. Those of skill in the art recognizethat a variety of techniques are available for constructing compoundswith the same or similar desired biological activity as the lead peptidecompound but with more favorable activity than the lead with respect tosolubility, stability, and susceptibility to hydrolysis and proteolysis.See Morgan and Gainor (1989) Ann. Rep. Med. Chem. 24:243–252,incorporated herein by reference. These techniques include replacing thepeptide backbone with a backbone composed of phosphonates, amidates,carbamates, sulfonamides, secondary amines, and N-methylamino acids.

Peptide mimetics wherein one or more of the peptidyl linkages [—C(O)NH—]have been replaced by such linkages as a —CH₂-carbamate linkage, aphosphonate linkage, a —CH₂-sulfonamide linkage, a urea linkage, asecondary amine (—CH₂NH—) linkage, and an alkylated peptidyl linkage[—C(O)NR₆— where R₆ is lower alkyl] are prepared during conventionalpeptide synthesis by merely substituting a suitably protected amino acidanalogue for the amino acid reagent at the appropriate point duringsynthesis.

Suitable reagents include, for example, amino acid analogues wherein thecarboxyl group of the amino acid has been replaced with a moietysuitable for forming one of the above linkages. For example, if onedesires to replace a —C(O)NR— linkage in the peptide with a—CH₂-carbamate linkage (—CH₂OC(O)NR—), then the carboxyl (—COOH) groupof a suitably protected amino acid is first reduced to the —CH₂OH groupwhich is then converted by conventional methods to a —OC(O)Clfunctionality or a para-nitrocarbonate —OC(O)O—C₆H₄-p-NO₂ functionality.Reaction of either of such functional groups with the free amine or analkylated amine on the N-terminus of the partially fabricated peptidefound on the solid support leads to the formation of a —CH₂OC(O)NR—linkage. For a more detailed description of the formation of such—CH₂-carbamate linkages, see Cho et al., Science 261:1303–1305 (1993).

Similarly, replacement of an amido linkage in the peptide with aphosphonate linkage can be achieved in the manner set forth in U.S. Pat.Nos. 5,359,115 and 5,420,328 to Campbell et al. and in U.S. patentapplication Ser. No. 08/081,577, the disclosures of which areincorporated herein by reference in their entirety.

Replacement of an amido linkage in the peptide with a —CH₂-sulfonamidelinkage can be achieved by reducing the carboxyl (—COOH) group of asuitably protected amino acid to the —CH₂OH group and the hydroxyl groupis then converted to a suitable leaving group such as a tosyl group byconventional methods. Reaction of the tosylated derivative with, forexample, thioacetic acid followed by hydrolysis and oxidativechlorination will provide for the —CH₂—S(O)₂Cl functional group whichreplaces the carboxyl group of the otherwise suitably protected aminoacid. Use of this suitably protected amino acid analogue in peptidesynthesis provides for inclusion of an —CH₂S(O)₂NR— linkage whichreplaces the amido linkage in the peptide thereby providing a peptidemimetic. For a more complete description on the conversion of thecarboxyl group of the amino acid to a —CH₂S(O)₂Cl group, see, forexample, Chemistry & Biochemistry of Amino Acids, Peptides and Proteins,Boris Weinstein (ed.), Vol. 7, pp. 267–357, Marcel Dekker, Inc., NewYork (1983) which is incorporated herein by reference.

Replacement of an amido linkage in the peptide with a urea linkage canbe achieved in the manner set forth in U.S. patent application Ser. No.08/147,805 which application is incorporated herein by reference in itsentirety.

Secondary amine linkages wherein a —CH₂NH— linkage replaces the amidolinkage in the peptide can be prepared by employing, for example, asuitably protected dipeptide analogue wherein the carbonyl bond of theamido linkage has been reduced to a CH₂ group by conventional methods.For example, in the case of diglycine, reduction of the amide to theamine will yield after deprotection H₂NCH₂CH₂NHCH₂COOH which is thenused in N-protected form in the next coupling reaction. The preparationof such analogues by reduction of the carbonyl group of the amidolinkage in the dipeptide is well known in the art.

The suitably protected amino acid analogue is employed in theconventional peptide synthesis in the same manner as would thecorresponding amino acid. For example, typically about 3 equivalents ofthe protected amino acid analogue are employed in this reaction. Aninert organic diluent such as methylene chloride or DMF is employed and,when an acid is generated as a reaction by-product, the reaction solventwill typically contain an excess amount of a tertiary amine to scavengethe acid generated during the reaction. One particularly preferredtertiary amine is diisopropylethylamine which is typically employed inabout 10 fold excess. The reaction results in incorporation into thepeptide mimetic of an amino acid analogue having a non-peptidyl linkage.Such substitution can be repeated as desired such that from zero to allof the amido bonds in the peptide have been replaced by non-amido bonds.

One can also cyclize the peptides of the invention, or incorporate adesamino or descarboxy residue at the termini of the peptide, so thatthere is no terminal amino or carboxyl group, to decrease susceptibilityto proteases or to restrict the conformation of the peptide. C-terminalfunctional groups of the compounds of the present invention includeamide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,and carboxy, and the lower ester derivatives thereof, and thepharmaceutically acceptable salts thereof.

V. Derivatives Having Disulfide Bonds

The compounds of the present invention may exist in a cyclized form withan intramolecular disulfide bond between the thiol groups of thecysteines. Alternatively, an intermolecular disulfide bond between thethiol groups of the cysteines can be produced to yield a dimeric (orhigher oligomeric) compound. One or more of the cysteine residues mayalso be substituted with a homocysteine. These intramolecular orintermolecular disulfide derivatives can be represented schematically asshown below:

wherein m and n are independently 1 or 2.

Other embodiments of this invention provide for analogs of thesedisulfide derivatives in which one of the sulfurs has been replaced by aCH₂ group or other isostere for sulfur. These analogs can be made via anintramolecular or intermolecular displacement, using methods known inthe art as shown below:

wherein p is 1 or 2. One of skill in the art will readily appreciatethat this displacement can also occur using other homologs of theα-amino-γ-butyric acid derivative shown above and homocysteine.

Alternatively, the amino-terminus of the peptide can be capped with analpha-substituted acetic acid, wherein the alpha substituent is aleaving group, such as an α-haloacetic acid, for example, α-chloroaceticacid, α-bromoacetic acid, or α-iodoacetic acid. The compounds of thepresent invention can be cyclized or dimerized via displacement of theleaving group by the sulfur of the cysteine or homocysteine residue.See, e.g., Barker et al., J. Med. Chem. 35:2040–2048 (1992) and Or etal., J. Org. Chem. 56:3146–3149 (1991), each of which is incorporatedherein by reference.

VI. Hydrophilic Polymer Addition

In addition to the foregoing N-terminal and C-terminal modifications,the compounds of the invention, including peptidomimetics, canadvantageously be modified with or covalently coupled to one or more ofa variety of hydrophilic polymers. The corresponding derivative may haveincreased solubility and circulation half-lives and maskedimmunogenicity. Nonproteinaceous polymers suitable for use in accordancewith the present invention include, but are not limited to,polyalkylethers as exemplified by polyethylene glycol and polypropyleneglycol; polylactic acid; polyglycolic acid; polyoxyalkenes;polyvinylalcohol; polyvinylpyrrolidone; cellulose and cellulosederivatives; dextran and dextran derivatives; etc. Generally, suchhydrophilic polymers have an average molecular weight ranging from about500 to about 100,000 daltons, more preferably from about 2000 to about40,000 daltons, and even more preferably, from about 5,000 to about20,000 daltons. In preferred embodiments, such hydrophilic polymers havean average molecular weight of about 5,000 daltons, 10,000 daltons, or20,000 daltons.

The compounds of the invention can be derivatized with or coupled tosuch polymers using any of the methods set forth in ZallipskyBioconjugate Chem. 6:150–165 (1995); Monfardini et al., BioconjugateChem. 6:62–69 (1995); U.S. Pat. No. 4,640,835; U.S. Pat. No. 4,496,689;U.S. Pat. No. 4,301,144; U.S. Pat. No. 4,670,417; U.S. Pat. No.4,791,192; U.S. Pat. No. 4,179,337 or WO 95/34326.

In a presently preferred embodiment, the compounds of the presentinvention are derivatized with polyethylene glycol (PEG). PEG is alinear, water-soluble polymer of ethylene oxide repeating units with twoterminal hydroxyl groups. PEGs are classified by their molecular weightswhich typically range from about 500 daltons to about 40,000 daltons. Ina presently preferred embodiment, the PEGs employed have molecularweights ranging from 5,000 daltons to about 20,000 daltons. PEGs coupledto the compounds of the present invention can be either branched orunbranched. See, e.g., Monfardini et al., Bioconjugate Chem. 6:62–69(1995). PEGs are commercially available from Shearwater Polymers Inc.(Huntsville, Ala.), Sigma Chemical Co., and other companies. Such PEGsinclude, but are not limited to, monomethyoxypolyethylene glycol(MePEG-OH); monomethoxypolyethylene glycol-succinate (MePEG-S);monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS);monomethoxypolyethylene glycol amine (MePEG-NH2);monomethoxypolyethylene glycol-tresylate (MePEG-TRES); andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

Briefly, in one exemplar embodiment, the hydrophilic polymer which isemployed, e.g., PEG, is preferably capped at one end by an unreactiveprotecting group, such as a methoxy or ethoxy. Thereafter, the polymeris activated at the other end by reaction with a suitable activatingagent, such as cyanuric halide (e.g., cyanuric chloride, bromide, orfluoride), diimadozole, an anhydride reagent (e.g., a dihalosuccinicanhydride, such as dibromosuccinic anhydride), acyl azide,p-diazoiumbenzyl ether), 3-(p-diazoniumphenoxy)-2-hydroxypropylether andthe like. The activated polymer is then reacted with a compound of thepresent invention to produce a compound derivatized with a polymer. Theamount of derivatization will be dependent, in part, upon theavailability of free amine groups in the compound of the presentinvention. For example, if all but one of the primary amine groups ofthe compound is protected, e.g., by an acyl group, a monoPEGylatedcompound will be formed. In a preferred embodiment of the invention, theamino terminus of a peptide or peptide mimetic compound is derivatizedwith PEG.

Alternatively, a functional group in the compounds of the invention canbe activated for reaction with the polymer, or the two groups can bejoined in a concerted coupling reaction using known coupling methods. Itwill be readily appreciated that the compounds of the invention can bederivatized with PEG using a myriad of other reaction schemes known toand used by those of skill in the art.

VII. Dimerization

One embodiment of this invention is drawn to dimers wherein side chainamino groups of each monomeric subunit are coupled via a urea linkage.Other embodiments are drawn to dimers wherein side chain amino groups ofeach monomeric subunit are coupled via disulfide, amide, or carbamatelinkages. The dimeric compounds may also contain intramolecular cysteinelinkages. Most preferably, the monomeric subunits will be dimerized toyield compounds having both intramolecular and intermolecular disulfidebonds. These dimers can be prepared as described below using techniquesknown to those of skill in the art and appropriate protecting groupstrategies.

In a preferred embodiment of the invention, a dimeric compound isderivatized with a hydrophilic polymer, such as PEG. One or both of themonomers may be derivatized with a hydrophilic polymer. In a morepreferred embodiment, the compound comprises a dimer with PEG covalentlybound to the amino termini of both of the monomers.

VIII. In Vivo and In Vitro Testing

The activity of the compounds of the present invention can be evaluatedin vivo in one of the numerous animal models of asthma. See Larson,“Experimental Models of Reversible Airway Obstruction,” in The Lung:Scientific Foundations, Crystal, West et al., eds., Raven Press, NewYork, 1991; Warner et al., Am. Rev. Respir. Dis. 141:253–257 (1990). Anideal animal model would duplicate the chief clinical and physiologicalfeatures of human asthma, including: airway hyperresponsiveness tochemical mediators and physical stimuli; reversal of airway obstructionby drugs useful in human asthma (β-adrenergics, methylxanthines,corticosteroids, and the like); airway inflammation with infiltration ofactivated leukocytes; and chronic inflammatory degenerative changes,such as basement membrane thickening, smooth muscle hypertrophy, andepithelial damage. Species used historically as animal models includemice, rats, guinea pigs, rabbits, dogs, and sheep. All have somelimitations, and the proper choice of animal model depends upon thequestion which is to be addressed.

The initial asthmatic response can be evaluated in guinea pigs, anddogs, and particularly, with a basenji-greyhound cross strain whichdevelops nonspecific airway hyperresponsiveness to numerousnonallergenic substances, such as methacholine and citric acid. Certainselected sheep exhibit a dual response after antigen challenge withAscaris proteins. In dual responding animals, the initial asthmaticresponse (IAR) is followed by a late asthmatic response (LAR) at 6–8hours post-exposure. Hypersensitivity to the cholinergic agonistcarbachol increases at 24 hours after antigen challenge in those animalswhich exhibit LAR.

The allergic sheep model can be used to evaluate the potentialantiasthmatic effects of the compounds of the present invention.Administration of compositions comprising aerosolized solutions of thecompounds of the instant invention to allergic sheep prior to orfollowing exposure to specific allergens will demonstrate that suchcompositions substantially lessen or abolish the late asthmatic responseand consequent hyperresponsiveness.

The compounds of this invention are also useful for the treatment ofother immunomediated inflammatory disorders in which tryptase activitycontributes to the pathological condition. Such diseases includeinflammatory diseases associated with mast cells, such as rheumatoidarthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis andother arthritic conditions, inflammatory bowel disease, peptic ulcer andvarious skin conditions.

The efficacy of the compounds of the instant invention for the treatmentof the vast majority of immunomediated inflammatory disorders can beevaluated by either in vitro or in vivo procedures. Thus, theanti-inflammatory efficacy of the compounds of the instant invention canbe demonstrated by assays well known in the art, for example, theReversed Passive Arthus Reaction (RPAR)-PAW technique (see, e.g.,Ganguly et al., U.S. Pat. No. 5,126,352, (1992)). Assays for determiningthe therapeutic value of compounds in the treatment of various skinconditions, such as hyperproliferative skin disease, are well known inthe art, for example, the Arachidonic Acid Mouse Ear Test (Id.). Thecompounds of the instant invention can be evaluated for their antiulceractivity according to the procedures described in Chiu et al., ArchivesInternationales de Pharmacodynamie et de Therapie 270:128–140 (1984).

IX. In Vitro Uses

The compounds of the invention are useful in vitro as unique tools forunderstanding the biological role of IL-5, including the evaluation ofthe many factors thought to influence, and be influenced by, theproduction of IL-5 and the receptor binding process. The presentcompounds are also useful in the development of other compounds thatbind to the IL-5R, because the present compounds provide importantinformation on the relationship between structure and activity thatshould facilitate such development.

The compounds are also useful as competitive inhibitors or tracers inassays to screen for new IL-5 receptor blockers. In such assayembodiments, the compounds of the invention can be used withoutmodification or can be modified in a variety of ways; for example, bylabeling, such as covalently or non-covalently joining a moiety whichdirectly or indirectly provides a detectable signal. In any of theseassays, the materials thereto can be labeled either directly orindirectly. Possibilities for direct labeling include label groups suchas: radiolabels such as [¹²⁵I], enzymes (U.S. Pat. No. 3,645,090) suchas peroxidase and alkaline phosphatase, and fluorescent labels (U.S.Pat. No. 3,940,475) capable of monitoring the change in fluorescenceintensity, wavelength shift, or fluorescence polarization. Possibilitiesfor indirect labeling include biotinylation of one constituent followedby binding to avidin coupled to one of the above label groups. Thecompounds may also include spacers or linkers in cases where thecompounds are to be attached to a solid support.

Thus, the compositions and methods of the present invention also can beused in vitro for testing a patient's susceptibility to varyingtreatment regimens for disorders associated with the overproduction ofIL-5 or an improper response to IL-5 using an in vitro diagnostic methodwhereby a specimen is taken from the patient and is treated with a IL-5Rbinding, IL-5 blocking compound of the present invention to determinethe effectiveness and amount of the compound necessary to produce thedesired effect. The blocking compound and dosage can be varied. Afterthe blocking compounds are screened, then the appropriate treatment anddosage can be selected by the physician and administered to the patientbased upon the results. Therefore, this invention also contemplates useof a blocking compound of this invention in a variety of diagnostic kitsand assay methods.

A further aspect of the invention is the use of compounds of the presentinventions for the manufacture of a medicament for the treatment and/orprevention of a variety of IL-5 disorders.

X. In Vivo Uses

The compounds of the invention can also be administered to warm bloodedanimals, including humans, to block the binding of IL-5 to the IL-5R invivo. Thus, the present invention encompasses methods for therapeutictreatment of IL-5 related disorders that comprise administering acompound of the invention in amounts sufficient to block or inhibit thebinding of IL-5 to the IL-5R in vivo. For example, the peptides andcompounds of the invention can be administered to treat symptoms relatedto the overproduction of IL-5 or an improper response to IL-5. Thecompositions and methods described herein will find use for thetreatment and/or prevention of a variety of IL-5 related disorders.

According to one embodiment, the compositions of the present inventionare useful for preventing or ameliorating asthma. In using thecompositions of the present invention in a treatment of asthma, thecompounds typically will be administered prophylactically prior toexposure to allergen or other precipitating factor, or after suchexposure. The compounds of the instant invention are particularly usefulin ameliorating the late-phase tissue destruction seen in both seasonaland perennial rhinitis. Another aspect of the present invention isdirected to the prevention and treatment of other immunomediatedinflammatory disorders associated with mast cells such as urticaria andangioedema, and eczematous dermatitis (atopic dermatitis), andanaphylaxis, as well as hyperproliferative skin disease, peptic ulcers,and the like.

Accordingly, the present invention also provides pharmaceuticalcompositions comprising, as an active ingredient, at least one of thepeptides or peptide mimetics of the invention in association with apharmaceutical carrier or diluent. The compounds of this invention canbe administered by oral, pulmonary, parenteral (intramuscular,intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation(via a fine powder formulation), transdermal, nasal, vaginal, rectal, orsublingual routes of administration and can be formulated in dosageforms appropriate for each route of administration.

Typically, when the compounds of the instant invention are to be used inthe treatment of asthma, they will be formulated as aerosols. The term“aerosol” includes any gas-borne suspended phase of the compounds of theinstant invention which is capable of being inhaled into the bronchiolesor nasal passages. Specifically, aerosol includes a gas-borne suspensionof droplets of the compounds of the instant invention, as may beproduced in a metered dose inhaler or nebulizer, or in a mist sprayer.Aerosol also includes a dry powder composition of a compound of theinstant invention suspended in air or other carrier gas, which may bedelivered by insufflation from an inhaler device, for example.

For solutions used in making aerosols of the present invention, thepreferred range of concentration of the compounds of the instantinvention is 0.1–100 milligrams (mg)/milliliter (mL), more preferably0.1–30 mg/mL, and most preferably, 1–10 mg/mL. Usually the solutions arebuffered with a physiologically compatible buffer such as phosphate orbicarbonate. The usual pH range is 5 to 9, preferably 6.5 to 7.8, andmore preferably 7.0 to 7.6. Typically, sodium chloride is added toadjust the osmolarity to the physiological range, preferably within 10%of isotonic.

Suspensions of the compounds of the present invention inhydrofluoronalkane propellants, especially 1,1,1,2-tetrafluoroethane of1,1,1,2,3,3,3-heptafluoropropane, optionally in the presence of asurfactant and/or cosolvent (e.g., ethanol) in a pressurized canistermay also be provided together with a suitable delivery device for thetreatment of the above mentioned respiratory disorders, especiallyasthma and allergic rhinitis.

Formulation of such solutions for creating aerosol inhalants isdiscussed in Remington's Pharmaceutical Sciences, see also, Gandertonand Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987);Gonda Critical Reviews in Therapeutic Drug Carrier Systems 6:273–313(1990); and Raeburn et al., J. Pharmacol. Toxicol. Methods 27:143–159(1992).

Solutions of the compounds of the instant invention may be convertedinto aerosols by any of the known means routinely used for makingaerosol inhalant pharmaceuticals. In general, such methods comprisepressurizing or providing a means of pressurizing a container of thesolution, usually with an inert carrier gas, and passing the pressurizedgas through a small orifice, thereby pulling droplets of the solutioninto the mouth and trachea of the animal to which the drug is to beadministered. Typically, a mouthpiece is fitted to the outlet of theorifice to facilitate delivery into the mouth and trachea.

In one embodiment, devices of the present invention comprise solutionsof the compounds of the instant invention connected to or containedwithin any of the conventional means for creating aerosols in asthmamedication, such as metered dose inhalers, jet nebulizers, or ultrasonicnebulizers. Optionally such device may include a mouthpiece fittedaround the orifice.

In an embodiment for the treatment of allergic rhinitis, a device maycomprise a solution of a compound of the instant invention in a nasalsprayer.

A dry powder comprising a compound of the instant invention, optionallywith an excipient, is another embodiment of the present invention. Thismay be administered by a drug powder inhaler containing the abovedescribed powder.

The compounds of the inventions can also be used in the treatment ofimmunomediated inflammatory skin conditions, such as urticaria andangioedema, eczematous dermatitis, and hyperproliferative skin disease,e.g., psoriasis, in mammals. As a result of the topical administrationof a compound of the present invention, a remission of the symptoms canbe expected. Thus, one affected by an immunomediated inflammatory skincondition can expect a decrease in scaling, erythema, size of theplaques, pruritus, and other symptoms associated with the skincondition. The dosage of medicament and the length of time required forsuccessfully treating each individual patient may vary, but thoseskilled in the art will be able to recognize these variations and adjustthe course of therapy accordingly.

Also included within the invention are preparations for topicalapplication to the skin comprising a compound of the present invention,typically in concentrations in the range of from about 0.001% to 10%,together with a non-toxic, pharmaceutically acceptable topical carrier.These topical preparations can be prepared by combining an activeingredient according to this invention with conventional pharmaceuticaldiluents and carriers commonly used in topical dry, liquid, cream andaerosol formulations. Ointment and creams may, for example, beformulated with an aqueous or oily base with the addition of suitablethickening and/or gelling agents. Such bases may include water and/or anoil such as liquid paraffin or a vegetable oil such as peanut oil orcastor oil. Thickening agents which may be used according to the natureof the base include soft paraffin, aluminum stearate, cetostearylalcohol, propylene glycol, polyethylene glycols, woolfat, hydrogenatedlanolin, beeswax, and the like.

Lotions may be formulated with an aqueous or oily base and will, ingeneral, also include one or more of the following: stabilizing agents,emulsifying agents, dispersing agents, suspending agents, thickeningagents, coloring agents, perfumes, and the like.

Powders may be formed with the aid of any suitable powder base, e.g.,talc, lactose, starch, and the like. Drops may be formulated with anaqueous base or non-aqueous base also comprising one or more dispersingagents, suspending agents, solubilizing agents, and the like.

The topical pharmaceutical compositions according to this invention mayalso include one or more preservatives or bacteriostatic agents, e.g.,methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol,benzalkonium chlorides, and the like. The topical pharmaceuticalcompositions also can contain other active ingredients such asantimicrobial agents, particularly antibiotics, anesthetics, analgesics,and antipruritic agents.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., lubricating agents such as magnesium stearate. In thecase of capsules, tablets, and pills, the dosage forms may also comprisebuffering agents. Tablets and pills can additionally be prepared withenteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, with the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized by, forexample, filtration through a bacteria retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured using sterile water, or some other sterile injectablemedium, immediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as cocoa butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients well known in the art.

It should, of course, be understood that the compositions and methods ofthis invention can be used in combination with other agents exhibitingthe ability to modulate IL-5 synthesis, release, and/or binding and withother agents for the treatment of immunomediated inflammatory disorders,and particularly asthma. β-Adrenergic agonists are especially useful inthese combinations, because they provide symptomatic relief of theinitial asthmatic response, whereas the compounds of the presentinvention provide relief for the late asthmatic response. Preferredβ-adrenergic agonists in these solutions include any of the usualβ-agonists employed for the relief of asthma, such as albuterol,terbutaline, formoterol, fanoterol, or prenaline.

Other agents useful in combination with the compounds of the instantinvention include anticholinergics, such as ipratropium bromide, andanti-inflammatory corticosteroids (adrenocortical steroids) such asbeclomethasone, triamcinolone, flurisolide, or dexamethasone.

The compositions containing the compounds can be administered forprophylactic and/or therapeutic treatments. In therapeutic applications,compositions are administered to a patient already suffering from adisease, as described above, in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications. Anamount adequate to accomplish this is defined as “therapeuticallyeffective dose.” Amounts effective for this use will depend on theseverity of the disease and the weight and general state of the patient.

In prophylactic applications, compositions containing the compounds ofthe invention are administered to a patient susceptible to or otherwiseat risk of a particular disease. Such an amount is defined to be a“prophylactically effective dose.” In this use, the precise amountsagain depend on the patient's state of health and weight.

The quantities of the IL-5 blocking compound necessary for effectivetherapy will depend upon many different factors, including means ofadministration, target site, physiological state of the patient, andother medicants administered. Thus, treatment dosages should be titratedto optimize safety and efficacy. Typically, dosages used in vitro mayprovide useful guidance in the amounts useful for in situ administrationof these reagents. Animal testing of effective doses for treatment ofparticular disorders will provide further predictive indication of humandosage. Various considerations are described, e.g., in Gilman et al.,(eds), Goodman and Gilman's: The Pharmacological Basis of Therapeutics,8th ed., Pergamon Press (1990); and Remington's Pharmaceutical Sciences,7th ed., Mack Publishing Co., Easton, Pa. (1985).

The peptides and peptide mimetics of this invention are effective intreating IL-5 mediated conditions when administered at a dosage range offrom about 0.001 mg to about 10 mg/kg of body weight per day. Thespecific dose employed is regulated by the particular condition beingtreated, the route of administration as well as by the judgement of theattending clinician depending upon factors such as the severity of thecondition, the age and general condition of the patient, and the like.

Many ligand-binding receptors are comprised of multiple subunits. Wherethe ligand binding and the signal transduction activities of thereceptor are found on different subunits, receptor activation may bereduced or abolished by compounds that interfere with the association ofthe ligand-binding subunits with the signal-transducing subunits. It isherein disclosed that compounds that bind with high affinity to two ormore ligand-binding subunits of heteromeric receptors are effectivereceptor antagonists. It has been discovered that providing amultivalent compound effective to bind with high affinity to two or moreligand-binding subunits of a receptor macromolecule is effective toantagonize the action of a ligand at a receptor. For example, the IL-5receptor comprises an a subunit and a 1 subunit, where the β subunit isthe signal-transducing subunit. Compounds binding to two IL-5 α subunitsare herein disclosed to be potent, highly specific IL-5 receptorantagonists. Compounds that bind with high affinity to two α subunits ofthe interleukin 3 (IL-3) receptor and compounds that bind with highaffinity to two α subunits of the granulocyte/macrophagecolony-stimulating factor (GM-CSF) receptor are likewise expected to bepotent, highly specific receptor antagonists. The protein gp130 is thecommon signaling subunit for the heteromeric receptors of a number ofdifferent ligands, such as interleukin 6 (IL-6), leukemia inhibitoryfactor (LIF), oncostatin M, interleukin 12 (IL-12), and ciliaryneurotrophic factor (CNTF). Compounds that dimerize the ligand-bindingsubunits of these receptors would be very effective antagonists. Theinterleukin 2 (IL-2) receptor is also a heteromeric receptor withdistinct ligand-binding and signaling subunits suitable for antagonismby compounds that bind with high affinity to two ligand-binding IL-2subunits.

A method for identifying compounds that dimerize to ligand-bindingreceptor subunits involves the following steps listed below. Firstly,peptide libraries are screened against the ligand-binding receptorsubunit, using suitable screening methods known in the art and asdisclosed above. Clones that compete with ligands for binding at thetarget receptor are identified in this screening step. Next, thoseclones with an odd number of cysteine residues are identified. Peptidesfrom the clones with an odd number of cysteine residues are thensynthesized, with the cysteine(s) in reduced form. These peptides arethen allowed to oxidize in aqueous solution to form peptide dimers.Finally, these dimeric peptides are tested for dimeric binding of thereceptor subunit.

Alternatively, combinatorial libraries of dimeric molecules can bescreened against the ligand-binding subunit. Compounds that compete withligand for binding should be tested to determine if the compoundsdimerize to receptor subunits.

A third method comprises developing reporter cell lines that expresschimeric receptors consisting of the extracellular ligand-binding domainof a homomeric receptor such as GM-CSF. An example of such a chimera isprovided above (IL-5αECD). Such reporter cells would contain a reportergene such as luciferase whose transcription is upregulated when thechimeric receptor is activated by a dimerized ligand. These cells wouldthen be used to screen libraries of dimeric molecules as in thepreceding step.

Although only preferred embodiments of the invention are specificallydescribed above, it will be appreciated that modifications andvariations of the invention are possible without departing from thespirit and intended scope of the invention.

All patents, patent applications, journal articles and other referencesmentioned herein, both infra and supra, are incorporated by reference intheir entireties.

In these examples and throughout this specification, the abbreviationsemployed have their generally accepted meanings, as follows:

-   -   ABI=Applied Biosystems Inc.    -   BHA=benzhydrylamine resin    -   BSA=bovine serum albumin    -   DMEM=Dulbecco's Minimal Essential Medium    -   DMEM/F 12=Dulbecco's Minimal Essential Medium/Hamm's F12 Medium    -   ECD=Extracellular domain    -   ESMS=Electrospray MS    -   ng/ml=nanogram/milliliter    -   min=minutes    -   μl=microliter    -   μl/mg=microliter/milligram    -   hr=hours    -   HMP=p-hydroxymethylphenoxymethyl polystyrene resin    -   HPLC=High Pressure Liquid Chromatography    -   HBTU=O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   HOBt=1-hydroxybenzotriazole    -   IL-5=Interleukin 5    -   IL-5Rα=the alpha chain subunit of the human IL-5 Receptor    -   IL-5RαECD=ECD of the human IL-5 receptor a chain    -   MALDI/MS=Matrix Assisted Laser Desorption Ionization MS    -   MS=Mass Spectrometry    -   PAL=5-(4-aminomethyl-3,5-dimethoxyphenoxy)valeric acid    -   PBS=phosphate buffered saline    -   RP-HPLC=Reverse Phase HPLC    -   Tris=Tris(hydroxymethyl)aminomethane    -   TNF=Tumor Necrosis Factor

EXAMPLE 1

Solid Phase Peptide Synthesis

Various peptides of the invention were synthesized using the Merrifieldsolid phase synthesis techniques (see Steward and Young, Solid PhasePeptide Synthesis, 2d. edition (Pierce Chemical, Rockford, Ill. (1984)and Merrifield (1963) J. Am. Chem. Soc. 85:2149) on an AppliedBiosystems Inc. Model 431A or 433A peptide synthesizer. Alternatively, aMilligen/Biosearch 9600 automated instrument could be used.

The peptides were assembled using standard protocols of the AppliedBiosystems Inc. (ABI) System Software version 1.01. Each coupling wasperformed for one-two hours with HBTU and HOBt. Double-couplings wereperformed at each step.

The resin used was HMP resin or PAL resin (Milligen/Biosearch), which isa cross-linked polystyrene resin with5-(4′-Fmoc-aminomethyl-3,5′-dimethyoxyphenoxy)valeric acid as a linker.Use of PAL resin results in a carboxyl terminal amide functionality uponcleavage of the peptide from the resin. Upon cleavage, the HMP resinproduces a carboxylic acid moiety at the C-terminus of the finalproduct. Most reagents, resins, and protected amino acids (free or onthe resin) were purchased from Millipore or Applied Biosystems Inc.

Trityl (Trt), and/or t-butyl (tBu) were utilized as protecting groupsfor the Cys residues. The Fmoc group was used for amino protectionduring the coupling procedure. Primary amine protection on amino acidswas achieved with Fmoc and side chain protection groups were t-butyl forserine, tyrosine, aspartic acid, glutamic acid, and threonine; Pmc(2,2,5,7,8-pentamethylchroma sulfonate) for arginine;N-t-butyloxycarbonyl for tryptophan, and lysine; and N-trityl forhistidine, asparagine, and glutamine. Side chain protecting groups arenot shown in the figures below for convenience.

Removal of the peptides from the resin and simultaneous deprotection ofthe side chain functions were achieved by treatment with reagent K (5%water, 5% phenol, 5% thioanisole, 2.5% 1,2-ethanedithiol, and 82.5%trifluoroacetic acid) or slight modifications of it. Alternatively, inthe synthesis of those peptides, with an amidated carboxyl terminus, thefully assembled peptide was cleaved with a mixture of 90%trifluoroacetic acid, 5% ethanedithiol, and 5% water, initially at 4°C., and gradually increasing to room temperature. The deprotectedpeptides were precipitated with diethyl ether.

In all cases, purification was by preparative, reverse-phase, highperformance liquid chromatography on a C₁₈ bonded silica gel column witha gradient of acetonitrile/water in 0.1% trifluoroacetic acid. Thehomogeneous peptides were characterized by MALDI/MS or electrospray massspectrometry and amino acid analysis when applicable.

EXAMPLE 2

Dimer Synthesis: Regioselective Cysteine Pairing

This technique calls for the use of two different Cys protecting groups.The first disulfide bond is formed by selectively removing the first setof protecting groups. The second disulfide bond is formed by removingthe second set of protecting groups from the peptide as shownschematically below.

Regioselective Cysteine Pairing-Peptides Containing Tryptophan:

As shown below, Acm was used as the protecting group for the first Cysand Trt was used for the second and third Cys residues. The procedure ofTamamura et al., Chem. Commun. 151 (1998) was used to effect thedimerization. The linear purified peptide (with 1 —S—S— and 1-Acm) (80mg, 0.043 millimole) and silver trifluoromethanesulfonate (1.09 gm, 100equiv.) is dissolved in TFA-anisole mixture (160 ml at 20:1 v/v) to givea concentration of 2 mg of peptide in 1 ml of TFA-anisole.

The reaction mixture is stirred for three hours at room temperature andfiltered into cold ether. The precipitate is further washed three timeswith cold ether. Then the dried crude peptide is suspended in DMSO-1 MHCl (80:20 v/v). The concentration is 2.5 mg/1 ml solution. Afterstirring overnight, the peptide is further purified by RP-HPLC. Thefractions are collected and further analyzed by analytical HPLC. Thepure fractions are pooled together, lyophilized and characterized by MS.The overall yield is 12% by weight.

Regioselective Cysteine Pairing-Peptides Containing Other thanTryptophan:

As shown below, tBu- was used as the protecting group for the first Cys;whereas, Trt was used for the second and third Cys residues. Theprocedure of Akaji et al., Tetrahedron Letters 33:1073–1076 (1992) wasutilized to effect the dimerization.

To the linear purified peptide with (1 —S—S— and 1-tBu) (10 mg, 0.0053millimole) is added diphenylsulfoxide (10.75 mg, 10 equiv.) and anisole(2 or 3 drops). The above mixture is dissolved in trifluoroacetic acid(3 ml). To this mixture is added, chlorotrimethylsilane (67.5 ml, 100equiv.). The solution is stirred for 90 minutes at room temperature. Thereaction mixture is filtered into cold ether and the precipitate isfurther washed three times. The crude peptide is further purified byRP-HPLC and checked by MS. The overall yield is 60% by weight.

EXAMPLE 3

Dimer Synthesis: Oxidative Folding of Cysteine

This technique is described further in Morder et al., Biopolymers(Peptide Science) 40:207–234 (1996). Unlike the regioselective cysteinepairing method, only one type of protecting group for the cysteine wasused. The linear peptide with 3 —SH has been oxidized to form a dimerdirectly as described below.

The linear peptide with 3 —SH is oxidized in Tris buffer, pH 7.8 for twodays. The course of the reaction is monitored by HPLC. The product canbe purified by RP-HPLC. After lyophilization, the stereochemistry isconfirmed by strong cation exchange column chromatography and by massspectrum. The disulfide bond pattern can be checked by tryptic digest.

EXAMPLE 4

Dimer Synthesis: Orthogonal Protecting Groups

A peptide, AF17362, with sequence TGGGDGYVCVEWARCPTCK (residues 36–50 ofSEQ ID NO: 8), was synthesized with three cysteine residues in the freesulfhydryl form:

When AF17362 was dissolved in 50 mM Tris buffer, pH 7.8 and assayedimmediately, it competed with [¹²⁵I] IL-5 for binding to IL-5Rα ECD withan IC₅₀ of 120 nM. However, when the peptide solution was left at roomtemperature for 10 days the IC₅₀ in the binding assay dropped to 550 pM.Concomitant with this change in binding affinity, the peptide'sretention time by C₁₈ RP-HPLC shifted (but remained a single peak), andmass spectroscopic (MS) analysis revealed that the peptide hadspontaneously formed a disulfide-linked dimer.

To determine the disulfide pattern of the peptide, the dimeric form ofAF17362 was digested with trypsin and analyzed by LC-electrospray-MS andMALDI-MS. The MS analysis of the tryptic digest revealed fragmentsconsistent with the presence of a single symmetrical dimer structurecontaining an interchain disulfide bond between the cysteines inposition 9 and intrachain disulfide bonds between the cysteines inpositions 15 and 18 on each chain:

The presence of a turn-inducing proline residue at position 16 wouldseem likely to favor the formation of such a C₁₅–C₁₈ disulfide bondabove other possible combinations, and kinetic analysis of thedimerization process by nuclear magnetic resonance spectroscopyconfirmed the formation of this intramolecular bond prior tointermolecular dimerization.

A dimer peptide was prepared synthetically using orthogonal protectinggroups. Synthesis was performed on HMP resin using an ABI peptidesynthesizer 431 A or 433A and Fmoc- chemistry. Each step was doublecoupled using HBTU/HOBt reagent. The acetamidomethyl (Acm-) protectinggroup was used for the Cys in position 9 and trityl (Trt-) for the Cysin positions 15 and 18. Other protecting groups were tBu- for Thr andTyr, -OtBu for Asp and Glu, Boc- for Trp and Pmc- for Arg. After thefinal coupling, the N-terminal Fmoc- group was removed. The peptide wascleaved and deprotected from the resin with TFA-DCM-anisole-2mercaptoethanol (89.7%-10%-0.1%-0.2%). The crude peptide was furtherpurified by C₁₈ RP-HPLC. The peptide was oxidized overnight in 30%DMSO-water at 1 mg/ml to form the intrachain disulfide bond [J. P. Tam,C.-R. Wu, W. Liu, J.-W. Zhang, J. Am. Chem. Soc. 113: 6657 (1991)]. Theinterchain disulfide bond was formed by using AgOTf-TFA followed byDMSO-aq.HCl (Tamamura et al., Chem Commun. 1: 151 (1998)). The productwas purified by C₁₈ RP-HPLC and confirmed by ESMS and MALDI/MS. Theresulting peptide, AF18748,

was indistinguishable from the dimeric form of AF17362 by all criteriatested (RP-HPLC, capillary electrophoresis, and tryptic digest) andcompeted with [¹²⁵I] IL-5 for binding to IL-5Rα ECD with essentially thesame potency IC₅₀ of 780 pM).

The ability of AF18748 to bind to the native IL-5R α/β complex on TF-1cells was assessed in a competition binding assay (as described inMcKinnon et al., J. Exp. Med 186: 121 (1997)). The binding of [¹²⁵I]IL-5 to TF-1 cells was inhibited by either unlabeled IL-5 (IC₅₀ 310±20pM) or AF18748 (IC₅₀ 400±40 pM), demonstrating that AF18748 can potentlyblock IL-5 binding to the native heterodimeric form of the receptor,exhibiting an affinity similar to the natural ligand (FIG. 1).

The functional activity of AF18748 was assessed in a human eosinophiladhesion assay (following the method of D. Fattah, et al, Cytokine 8:248(1996)). The peptide alone was devoid of agonist activity, butcompletely inhibited IL-5 induced eosinophil adhesion to immobilized IgGwith an IC₅₀ of 348±67 pM (FIG. 2). Furthermore, concentrations ofAF18748 up to 1 μM had no effect on eosinophil adhesion induced by therelated cytokines GM-CSF and IL-3, the unrelated cytokine TNFα or thechemotactic peptide fMet-Leu-Phe (FIG. 3). AF18748 is therefore a potentand selective antagonist of IL-5 in a human eosinophil functional assay.

EXAMPLE 5

Bioassays

Bioactivity of synthetic peptides and MBP-peptide fusions is measuredusing a Cytosensor microphysiometer (Molecular Devices) to record themetabolic response of TF-1 cells (a human leukemia cell line) to IL-5 inthe presence or absence of peptide. After overnight incubation withoutIL-5, these cells exhibited a robust increase in metabolic activity whenIL-5 is added to the medium. This increase was measured by themicrophysiometer as an increase in the rate of acidification of weaklybuffered tissue culture medium.

TF-1 cells were seeded into microphysiometer chambers at a density of1.5×10⁵ cells/chamber and grown overnight in DMEM tissue culture mediumcontaining 10% fetal bovine serum, but lacking the 1 ng/ml IL-5 (R&DSystems) that is required for long-term maintenance of these cells inculture. The chambers were then placed in the microphysiometer andincubated with weakly buffered DMEM/F12 medium containing 1% human serumalbumin until a baseline rate of medium acidification was established.Varying dilutions of test peptide were then introduced for 15 min. Noneof the peptides tested had any effect on the baseline acidificationrate. IL-5 at 10 ng/ml was then introduced for 25 minutes in thecontinued presence of test peptide. The chambers were then flushed withfresh medium.

Typically, maximal response to IL-5 occurred within 20 min. of the onsetof IL-5 addition to the medium. In the absence of test peptide thisresponse was typically a 1.5 to 2-fold increase in the rate of mediumacidification. All peptides tested were able to reduce or completelyblock the response of the TF-1 cells to IL-5. Other, randomly chosencontrol peptides, at the same or higher concentrations, had no effect.The test peptides also had no effect on the robust microphysiometerresponse of TF-1 cells to TNFα, indicating that the test peptides wereexhibiting their effect by specifically antagonizing IL-5 action. TheIC₅₀ for test peptides was defined as that peptide concentration whichgave a 50% reduction in the maximal IL-5 response when compared to theresponse to IL-5 alone.

EXAMPLE 6

Binding Affinity

Binding affinities of synthetic peptides for IL-5Rα were measured in acompetition binding assay using radio-iodinated IL-5. Immulon 4(Dynatech) microtiter wells were coated with streptavidin (Sigma) byincubating 100 μl of a 50 μg/ml solution in PBS for 30 min. at 37°. Thewells were blocked with 200 μl of 1% BSA in PBS for 15 min. at 37°,followed by 100 μl of biotinylated monoclonal antibody, designated mAb179, at 5 μg/ml in PBS. Soluble IL-5Rα was then immobilized in the wellsby incubating 100 μl of a solution of soluble receptor harvest diluted1:5000 in PBS/0.1% BSA for 1 hr. at 4°. After washing away unboundreceptor, 50 μl of various concentrations of test peptide diluted inPBS/0.1% BSA were added to the wells, followed by 50 μl of a fixedconcentration of [¹²⁵I] IL-5 (Amersham). The binding reactions wereincubated at 4° C. for 2 hr, then washed with PBS to remove unbound[¹²⁵I] IL-5. Bound [¹²⁵I] IL-5 was determined by gamma counting. Totalbinding was defined by the amount of [¹²⁵I] IL-5 bound in the absence ofany competitor. Non-specific binding was defined by the amount of [¹²⁵I]IL-5 bound in the presence of 30 nM IL-5. Peptide binding data wasanalyzed to determine the peptide concentration required to reducespecific [¹²⁵I] IL-5 binding by 50% (IC₅₀). Under the conditionsdescribed the IC₅₀ values determined should be similar to thedissociation constant (K_(d)) of the peptides for IL-5Rα.

EXAMPLE 7

Scintillation Proximity Binding Assay

Binding affinities of synthetic peptides for IL-5Rα were measured in ascintillation proximity assay (SPA) using radio-iodinated IL-5.Streptavidin coated SPA beads (Amersham) were suspended in IL-5 bindingbuffer (phosphate buffered saline, 0.1% bovine serum albumin, 0.2% NaN₃)at 2 mg beads/ml of buffer. Biotinylated mAb 179 was adsorbed onto thebeads at a ratio of 2 μg Ab/mg beads by incubating the beads andantibody with agitation for at least 2 h. at 4°. The beads were pelletedat 2500 rpm and resuspended at 2 mg/ml in binding buffer. Soluble IL-5Rαwas then adsorbed onto the beads by diluting a solution of solublereceptor harvest 1:500 into the bead suspension and incubating withagitation for 2 hr at 4°. The beads were again pelleted at 2500 rpm andresuspended at 2 mg/ml in binding buffer. Competition binding assayswere carried out in 100 μl reactions in white polystyrene microtiterplates. These reactions contained 0.05 mg of receptor coated SPA beads,various concentrations of test peptide diluted in binding buffer, and 20pM [¹²⁵I] IL-5 (Amersham). The plates were incubated at room temperaturefor 1 hour with agitation, then centrifuged for 5 min at 1500 rpmfollowed by scintillation counting on a Topcount instrument (Packard).Total binding was defined by the amount of [¹²⁵I] IL-5 bound in theabsence of any competitor. Non-specific binding was defined by theamount of [¹²⁵I] IL-5 bound in the presence of 30 nM IL-5. Peptidebinding data was analyzed to determine the peptide concentrationrequired to reduce specific [¹²⁵I] IL-5 binding by 50% (IC₅₀). Resultsfor dimers of the following monomeric structures are provided below. Ineach instance, the dimer has both intramolecular and intermoleculardisulfide linkages as shown generically below:

SPA Microphysio- SEQ ID NO: Monomer Structure pIC₅₀ meter pIC₅₀ residues32–50 of SEQ ID NO: 8 (H)-TGGGDGYVCVEWARCPTCK-(OH) 9.1 9.6 residues36–50 of SEQ ID NO: 11 (H)-EGYVCVEWARCPTCK-(OH) 8.7 9.2 residues 36–50of SEQ ID NO: 12 (H)-EGYVCVEWARCPTCR-(OH) 8.4 9.6 residues 36–50 of SEQID NO: 14 (H)-EGYVCVEFARCPTCR-(OH) 6.8 N.D. residues 36–50 of SEQ ID NO:14 (H)-EGYVCVEFARCPTCR-(NH₂) 6.4 N.D. residues 37–50 of SEQ ID NO: 17(H)-GYVCVEFARCPTCG-(NH₂) 6.1 N.D. residues 36–50 of SEQ ID NO: 13(H)-EGYVCVEFARCPTCG-(NH₂) 6.6 N.D. residues 37–50 of SEQ ID NO: 18(H)-GYVCVEWARCPTCR-(OH) 8.2 8.4 residues 36–50 of SEQ ID NO: 12(H)-EGYVCVEWARCPTCR-(NH₂) 8.1 9.1 residues 36–50 of SEQ ID NO: 15(H)-EGYVCVEWARCPTCG-(NH₂) N.D. 9.1 residues 36–50 of SEQ ID NO: 12(Ac)-EGYVCVEWARCPTCR-(NH₂) N.D. 8.4 residues 36–50 of SEQ ID NO: 11(Ac)-EGYVCVEWARCPTCK-(NH₂) N.D. 8.5 residues 36–50 of SEQ ID NO: 12(Ahx)-EGYVCVEWARCPTCR-(NH₂) N.D. 9.0 residues 36–50 of SEQ ID NO: 10(Ac)-EGYVCVEWAACPTCR-(NH₂) N.D. 6.9 residues 36–50 of SEQ ID NO:9       residues 36–50 of SEQ ID NO: 9

N.D. 6.5

EXAMPLE 8

Biometric Imaging Assay

A “sandwich” binding assay using microvolume fluorimetry (Martens etal., Anal. Biochem., 273:20 (1999)) was used to measure the liganddependent association of two receptor proteins. IL-5RαECD (theextracellular domain (ECD) of the human IL-5 receptor a chain) wasimmobilized onto 10 μm beads and incubated with soluble, fluorescentlylabeled IL-5Rα ECD in the presence or absence of IL-5 or peptide.

Fluorescently labeled mAb 179 was prepared using Cy5 monofunctionalreactive dye (Amersham) according to the manufacturer's instructions.(As disclosed in Martens et al., supra, Cy5.5 dye is also suitable.)Fluorescent mAb179:IL-5Rα ECD complexes were formed by preincubating thelabeled antibody with an equimolar amount of receptor ECD in phosphatebuffered saline (PBS) for 1 hr at room temperature. Mono A 10 μmpolystyrene beads (Pharmacia) were coated first with biotinylated bovineserum albumin, then sequentially with streptavidin, biotinylated mAb179,and IL-5Rα ECD. The receptor coated beads were incubated in 100 μl PBScontaining 1% BSA, 0.02% sodium azide, and 0.05% Tween-20, 5 nM labeledmAb179:IL-5Rα ECD complex and serial dilutions of human IL-5, AF18748 (adimer of residues 32–50 of SEQ ID NO: 8), or AF 17121, a peptide monomerwith the sequence VDECWRIIASHTWFCAEE (residues 33–50 of SEQ ID NO: 19)with intramolecular disulfide linkages:

As shown in FIG. 4, fluorescently labeled IL-5Rα ECD was incubated withIL-5RαECD-coated polystyrene beads and serial dilutions of AF18748 (opencircles), AF17121 (closed circles), or human IL-5 (open squares). Thebinding reactions were incubated in microtiter plates overnight at roomtemperature, then microvolume fluorimetry measurements ofbead-associated fluorescence were made using an FMAT instrument(Perkin-Elmer). The data points represent averages of triplicatedeterminations from a single representative experiment, providingbiochemical evidence of AF 18748-induced IL-5-α dimerization.

Addition of increasing amounts of AF18748 over a range from 10 pM to 1nM caused an increase in the association of the fluorescently labeledreceptor to the beads (FIG. 4), indicating that the peptide is bindingsimultaneously to both an immobilized receptor and a soluble receptor.At concentrations of AF18748 greater than 1 nM the binding of labeledreceptor to the beads decreases, presumably due to the excess of peptidefavoring a 1:1 association of peptide with both immobilized and solublereceptor. Addition of the monomeric IL-5 antagonist peptide, AF17121,did not mediate the association of labeled receptor with the beads (FIG.4), demonstrating monovalent receptor binding. Moreover, although IL-5is a disulfide linked homodimer it also did not mediate receptorassociation. This is consistent with previous reports demonstrating thateach IL-5 dimer binds to a single IL-5Rα ECD in solution (R. Devos, etal, Journal Of Biological Chemistry 268:6581 (1993); K. Johanson, et al,J. Biol. Chem. 270:9459 (1995)).

Secondly, the ability of peptides to induce receptor dimerization wasstudied using gel filtration chromatography. Gel filtrationchromatography was performed on a Pharmacia Smart system using aPrecision Superdex 200 3.2/30 column at 25° C. with a flow rate of 10μl/min. The buffer was 50 mM Tris pH 7.5, 0.5 M NaCl. The column wascalibrated using the Biorad Gel filtration molecular weight standardswhich contain Thyroglobulin, 670 KD; Bovine gamma globulin, 158 KD;Chicken ovalbumin, 44 KD; Equine myoglobin, 17 KD; Vitamin B12, 1.35 KD.As shown in FIG. 5, IL-5Rα ECD was loaded onto the column alone (dashedline) or in the presence of equimolar amounts of AF17121 (dotted line)or AF18748 (solid line). Elution volumes and calculated molecularweights are given in Table 3. Results shown are from a singleexperiment, representative of three separate runs for each condition.

Soluble IL-5Rα ECD appeared as a single peak with a calculated molecularweight of 89.5 kDa (FIG. 5). Although this is considerably higher thanpredicted, the monomeric nature of the soluble receptor was confirmed bycomparing its behavior under native and denaturing (6M guanidinehydrochloride) conditions. The IL-5Rα is extensively glycosylated, andthis is probably the source of the discrepancy in molecular weight.

The effect of peptide monomer AF17121 was compared to the effect of thedimer AF18748. The presence of AF17121 had no effect on the elution ofthe receptor (FIG. 5). In contrast, the apparent size of the IL-5Rα ECDincreased dramatically in the presence of AF18748, consistent with achange from a monomeric to a dimeric form (FIG. 5, Table 3).

Thirdly, analytical ultracentrifugation was used to examine thestoichiometry of the AF18748:IL-5Rα ECD interaction. Velocitysedimentation experiments were conducted on a Beckman XLIUltracentrifuge equipped with absorption and interference optics and anAn60Ti rotor. The experiments were run at 40,000 rpm in charcoal-filledEpon double sector centerpieces and the data was collected at 280 nm assingle scans at a spacing of 0.01 cm in continuous scan mode. Thetemperature was held at 25° C. and the buffer was 50 mM Tris pH 7.5, 0.5M NaCl. For experiments with peptide, equal amounts were added to bothreference and sample compartments. Data was analyzed using bothUltrascan (Borroes Demeler) and DCDT (Walter Stafford). Data shown isfrom a single experiment representative of two others.

Analysis of data collected in velocity sedimentation experiments withthe IL-5Rα ECD revealed a Gaussian distribution of species centered at3.3S (FIG. 6). Addition of a molar equivalent of AF18748 resulted in achange in the shape of the distribution of species. In the presence of amolar equivalent of AF18748, analysis of the data revealed the presenceof two principal species, each with a Gaussian distribution, givingapparent sedimentation coefficient values of 3.2 S and 5.1 S. Theseresults are entirely consistent with the result expected to be obtainedwith peptide-induced receptor dimerization (FIG. 6).

TABLE 3 Elution Calculated Receptor Volume MW State AF17121 alone 2.0202.5 n.a. AF18748 alone 1.956 3.9 n.a. IL-5RαECD alone 1.427 89.5 MonomerIL-5RαECD + AF17121 1.425 89.5 Monomer 2.020 2.5 IL-5RαECD + AF187481.282 208.5 Dimer 1.425 89.5 Monomer 1.956 3.9

EXAMPLE 9

Chimeric IL-5Rα/EGFR Reporter Cell Assay

A system comprising a chimeric IL-5R-α/EGFR reported cell assay wasdesigned to analyze receptor binding stoichiometry in a cellularcontext. A chimeric receptor consisting of the IL-5Rα ECD fused to thetransmembrane spanning and intracellular domains of the epidermal growthfactor receptor (EGFR) was constructed, and stably expressed in Ba/F3cells that also contained a luciferase reporter gene under thetranscriptional control of the c-fos enhancer and minimal thymidinekinase promoter.

In the assay, Ba/F3 cells expressing a chimeric IL-5Rα/EGFR receptor andcontaining a luciferase reporter gene were starved overnight of the WEHIconditioned medium in which they were maintained, then seeded intomicrotiter wells at a density of 10⁵ cells/well. Following stimulation,the cells were incubated for 4 hr at 37° in a 5% CO₂ incubator. LucLitereagent (Packard) was then added to the wells according to themanufacturer's instructions and the plates were assayed for luciferaseactivity by counting in a Topcount instrument (Packard). Data pointsrepresent mean±SEM for 3 separate experiments performed in triplicate.The data has been normalized to the stimulation induced by 1 μM AF18748in each experiment. The experiments shown in FIG. 7, with cells treatedwith serial dilutions of AF18748 (closed circles), AF17121 (opencircles) or IL5 (open squares), demonstrate that AF18748 activates theIL-5Rα/EGFR chimeric receptor.

Since activation of native EGFR by ligand-induced homodimerizationresults in an increase in c-fos transcription (Hazzalin et al., Oncogene15:2321 (1997)), homodimerization of the IL-5Rα/EGFR chimera shouldlikewise induce expression of the luciferase transgene. Stimulation ofBa/F3 cells expressing the chimera with AF18748 resulted in a dosedependent increase in luciferase activity while the monovalent ligandsIL5 or AF17121 had no effect (FIG. 7).

Furthermore, activation of the luciferase reporter by AF18748 wasantagonized by IL-5 (FIG. 8), demonstrating that this is a specificIL-5-receptor mediated event. In these experiments, cells were treatedwith 200 pM AF18748 in the presence of increasing concentrations ofIL-5. Data points represent the average of triplicate determinationsfrom a single representative experiment, and demonstrate that IL-5inhibits the AF18748-induced activation of the chimeric IL-5Rα/EGFR inthis reporter cell assay.

This also demonstrates that the monovalent nature of the IL-5:IL-5Rαinteraction observed in solution (Devos et al., J. Biol. Chem 268:6851(1993); K. Johanson et al., J. Biol. Chem. 270:9459 (1995)) ismaintained when the receptors are anchored in a cell membrane. Treatmentof unstimulated cells with IL-5 or AF17121 actually reduced basal levelof luciferase expression (FIG. 7), and IL-5 reduced the AF18748stimulated level to well below control levels (FIG. 8). This suggeststhat there is some degree of ligand independent receptor dimerizationdue to overexpression, that can be inhibited by the binding of amonovalent ligand.

This combination of biochemical, biophysical and cell-based data showsthat AF18748 can bind to two IL-5Rα chains simultaneously. In contrastto the receptors for EPO and TPO where ligand induced receptorhomodimerization is required for activation, activation of the IL-5receptor involves the heterodimerization of the ligand binding α-chainwith the β_(c) signaling chain. Thus, while the dimeric nature of thepeptides that bind to the EPO and TPO receptors underpins their agonistactivity, the ability to occupy two IL-5 receptor α-chains leads to thehigh potency functional antagonism exhibited by AF18748. The multivalentbinding of the dimer peptide will sequester α-chains and thereby preventthe ligand-induced receptor heterodimerization required to initiatesignal transduction.

EXAMPLE 10

In Vivo Serum Stability

The length of time that peptide dimers remain in circulation wasmeasured by injection of peptides into mice and subsequently measuringthe amount of peptide remaining in circulation at various times afterinjection. As illustrated in FIG. 9, three peptide dimers with the samepeptide sequence but differing in the amount of PEGylation wereinvestigated. The compounds used were dimers comprising the peptidesequence SEQ ID NO: 8; the peptide dimer AF18748 (lacking PEG), themono-PEGylated peptide dimer AF25123, and the diPEGylated peptide dimerAF25122, where PEGylation was with 20 kDa PEG linked to the N-terminusof one or both peptides of a dimer.

Peptides were dissolved at 0.125 mM (AF18748) or 0.5 mM (AF25122,AF25123) in 0.9% (w/v) saline and dosed into individual female CD-1 mice(about 25 g) via a lateral tail vein (100 microliter injection). Atindicated times, shown in FIG. 9, terminal blood samples (2 animals pertimepoint) were collected, by cardiac puncture, into heparinized tubesand centrifuged to yield plasma which was stored frozen. Peptides wereextracted from the plasma by the addition of an equal volume of ice-coldacetonitrile and, following centrifugation, the supernatants containingthe peptides were dried down and stored at 4° C.

The amount of peptide present in the serum extracts was evaluated bysurface plasmon resonance techniques using a competition binding assayformat on a BIAcore 1000 machine (Biacore AB, Uppsala, Sweden). Adisulfide-linked dimer peptide of the sequence EGYVCVEWARCPTCK (residues36–50 of SEQ ID NO: 11) was chemically coupled onto a CM-5 Biosensorchip using the manufacturer's standard protocol. Purified recombinantIL-5 receptor alpha chain extracellular domain was passed over thepeptide coated chip and the binding of the IL-5 receptor to theEGYVCVEWARCPTCK (residues 36–50 SEQ ID NO: 11) peptide dimer wasmonitored. The binding of IL-5 receptor alpha extracellular domain tothe peptide coated surface could be inhibited in a dose dependent mannerby each of the IL-5 antagonist peptides in solution. Thus the amount ofinhibitory activity present in the serum extracts can be compared to astandard curve of known amounts of the same peptide to allow an accuratedetermination of the amount of peptide in the blood.

1. A compound comprising a sequence of amino acids selected from thegroups consisting of TGGGDGYVX₃VE X₄ ARCPTCK (residues 32–50 of SEQ IDNO: 1); EGYVX₃VE X₄ ARCPTCK (residues 36–50 of SEQ ID NO: 2); EGYVX₃VEX₄ ARCPTCR (residues 36–50 of SEQ ID NO: 3); GYVX₃VE X₄ ARCPTCG(residues 36–50 of SEQ ID NO: 4); EGYVX₃VE X₄ ARCPTCG (residues 36–50 ofSEQ ID NO: 5); GYVX₃VE X₄ ARCPTCR (residues 37–50 of SEQ ID NO: 6); andEGYVX₃VE X₄ AACPTCR (residues 36–50 of SEQ ID NO: 7) wherein X₃ is Cys,Lys, or Dpr, and X₄ is Nal, Trp, or Phe.
 2. The compound of claim 1,wherein the sequence is fourteen to fifty amino acid residues in length.3. The compound of claim 2, wherein the sequence is fourteen to twentyamino acid residues in length.
 4. A compound of claim 2 or claim 3,wherein the N-terminus of said sequence is selected from the groupconsisting of —NRR, —NRC(O)R, —NRC(O)OR, NRS (O)₂R, —NHC(O)NHR,succinimide, benzyloxycarbonyl-NH—, and benzyloxycarbonyl-NH— havingfrom 1 to 3 substituents on the phenyl ring of saidbenzyloxycarbonyl-NH—, said benzyloxycarbonyl-NH— substituents selectedfrom the group consisting of lower alkyl, lower alkoxy, chloro, andbromo, where R and R, are independently selected from the groupconsisting of hydrogen and lower alkyl, wherein the C-terminus of saidsequence has the formula —C(O)R₂ where R₂ is selected from the groupconsisting of hydroxy, lower alkoxy, and —NR₃R₄ wherein R₃ and R₄ areindependently selected from the group consisting of hydrogen and loweralkyl and wherein the nitrogen atom of the —NR₃R₄ group can optionallybe the amine group of the N-terminus of the peptide so as to form acyclic peptide, and physiologically acceptable salts thereof.
 5. Thecompound of claim 1, wherein the compound binds to the interleukin 5receptor (IL-5R) with an IC₅₀ of no more than about 2 mM as determinedby a binding affinity assay.
 6. The compound of claim 1, wherein thecompound binds to the IL-5R with an IC₅₀ of no more than about 100 uM asdetermined by a binding affinity assay.
 7. A compound comprising asequence of amino acids selected from the groups consisting ofX₁-TGGGDGYVX₃VEX₄ARCPTCK-X₂ (residues 32–50 of SEQ ID NO: 1);X₁-EGYVX₃VEX₄ARCPTCK-X₂ (residues 36–50 of SEQ ID NO: 2);X₁-EGYVX₃VEX₄ARCPTCR-X₂ (residues 36–50 of SEQ ID NO: 3);X₁-GYVX₃VEX₄ARCPTCG-X₂ (residues 36–50 of SEQ ID NO: 4);X₁-EGYVX₃VEX₄ARCPTCG-X₂ (residues 36–50 of SEQ ID NO: 5);X₁-GYVX₃VEX₄ARCPTCR-X₂ (residues 37–50 of SEQ ID NO: 6); andX₁-EGYVX₃VEX₄AACPTCR-X₂ (residues 36–50 of SEQ ID NO: 7) wherein X₁ ishydrogen or acyl; X₂ is —NH₂ or —OH wherein —NH₂ indicates that thecarboxy terminus of the compound has been amidated and —OH indicatesthat the carboxy terminus of the compounds has not been derivatized; X₃is Cys, Lys, or Dpr, and X₄ is Nal, Trp, or Phe.
 8. The compound ofclaim 7, wherein the compound binds to the IL-5R with an IC₅₀ of no morethan about 2 mM as determined by a binding affinity assay.
 9. Thecompound of claim 7, wherein the compound binds to the IL-5R with anIC₅₀ of no more than about 100 uM as determined by a binding affinityassay.
 10. A compound comprising an amino acid sequence selected fromthe group consisting of (H)-TGGGDGYVCVEWARCPTCK-(OH) (residues 32–50 ofSEQ ID NO: 8); (Ac)-EGYV(Dpr)VEWARCPTCR-(NH₂) (residues 36–50 of SEQ IDNO: 9); (Ac)-EGYVCVEWAACPTCR-(NH₂) (residues 36–50 of SEQ ID NO: 10);(Ac)-EGYVCVEWARCPTCK-(NH₂) (residues 36–50 of SEQ ID NO: 11);(Ac)-EGYVCVEWARCPTCK-(OH) (residues 36–50 of SEQ ID NO: 11);(Ac)-EGYVCVEWARCPTCR-(NH2) (residues 36–50 of SEQ ID NO: 12);(Ahx)-EGYVCVEWARCPTCR-(NH2) (residues 36–50 of SEQ ID NO: 12);(H)-EGYVCVEFARCPTCG-(NH2) (residues 36–50 of SEQ ID NO: 13);(H)-EGYVCVEFARCPTCR-(OH) (residues 36–50 of SEQ ID NO: 14);(H)-EGYVCVEWARCPTCG-(NH2) (residues 36–50 of SEQ ID NO: 15);(H)-EGYVCVEWARCPTCK-(NH2) (residues 36–50 of SEQ ID NO: 11);(H)-EGYVCVEWARCPTCK-(OH) (residues 36–50 of SEQ ID NO: 11);(Ac)-EGYVCVEWARCPTCR-(OH) (residues 36–50 of SEQ ID NO: 12);(H)-EGYVCVEWARCPTCR-(NH2) (residues 36–50 of SEQ ID NO: 12);(H)-EGYVCVEWARCPTCR-(OH) (residues 36–50 of SEQ ID NO: 12);(H)-EGYVKVEWARCPTCR-(OH) (residues 36–50 of SEQ ID NO: 16);(H)-GYVCVEFARCPTCG-(NH2) (residues 37–50 of SEQ ID NO: 17); and(H)-GYVCVEWARCPTCR-(OH) (residues 37–50 of SEQ ID NO: 18) wherein —(NH₂)indicates that the carboxy terminus of the compound has been amidated,—(OH) indicates that the carboxy terminus of the compound has not beenderivatized, (Ac) indicates that the amino terminus of the compound hasbeen acetylated, and (Ahx)- indicates that the amino terminus of thecompound has been acylated with aminohexanoic acid.
 11. The compound ofclaim 10, wherein the compound binds to the IL-5R with an IC₅₀ of nomore than about 2 mM as determined by a binding affinity assay.
 12. Thecompound of claim 10, wherein the compound binds to the IL-5R with anIC₅₀ of no more than about 100 uM as determined by a binding affinityassay.
 13. The compound of claim 1, wherein the compound furthercomprises an intramolecular disulfide linkage between two Cys residues.14. The compound of claim 1, wherein the compound further comprises adimer with intermolecular disulfide, amide, carbamate, or urea linkages.15. The compound of claim 1, wherein the compound is coupled to apolyethylene glycol molecule.
 16. The compound of claim 15, wherein thepolyethylene glycol has an average molecular weight of between about 500to about 100,000 daltons.
 17. The compound of claim 15, wherein thepolyethylene glycol has an average molecular weight of between about2,000 to about 40,000 daltons.
 18. A compound of claim 15, wherein thepolyethylene glycol has an average molecular weight of between about5,000 to about 20,000 daltons.
 19. A pharmaceutical compositioncomprising a therapeutically effective amount of a compound of claim 1in combination with a pharmaceutically acceptable carrier.
 20. Apharmaceutical composition comprising a therapeutically effective amountof a compound of claim 7 in combination with a pharmaceuticallyacceptable carrier.
 21. A pharmaceutical composition comprising atherapeutically effective amount of a compound of claim 10 incombination with a pharmaceutically acceptable carrier.
 22. A method fortreating a patient having a disorder that is susceptible to treatmentwith an IL-5 inhibitor, comprising the steps of providing atherapeutically effective amount of a compound of claim 1, andadministering said therapeutically effective amount of compound to thepatient.
 23. A method for treating a patient having a disorder that issusceptible to treatment with an IL-5 inhibitor, comprising the steps ofproviding a therapeutically effective amount of a compound of claim 7,and administering said therapeutically effective amount of compound tothe patient.
 24. A method for treating a patient having a disorder thatis susceptible to treatment with an IL-5 inhibitor, comprising the stepsof providing a therapeutically effective amount of a compound of claim10, and administering said therapeutically effective amount of compoundto the patient.